KETOHEXOKINASE (KHK) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the ketohexokinase (KHK) gene, and methods of using such RNAi agents to inhibit expression of KHK and methods of treating subjects having a KHK-associated disorder, e.g., liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), disorders of glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney disorder, tubular dysfunction, proinflammatory changes to the proximal tubules), metabolic syndrome, adipocyte dysfunction, visceral adipose deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/445,294, filed on Jun. 19, 2019, which is a divisional application ofU.S. patent application Ser. No. 15/224,805, filed on Aug. 1, 2016, nowU.S. Pat. No. 10,370,666, issued on Aug. 6, 2019, which is a 35 § U.S.C.111(a) continuation application which claims the benefit of priority toPCT/US2015/015367, filed on Feb. 11, 2015, which in turn claims priorityto U.S. Provisional Patent Application No. 61/938,567, filed on Feb. 11,2014. The entire contents of each of the foregoing applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 5, 2021, isnamed 121301_01204_SL.txt and is 116,326 bytes in size.

BACKGROUND OF THE INVENTION

Epidemiological studies have shown that the western diet is one of theleading causes of the modern obesity pandemic. Increase in fructoseuptake, associated with the use of enriched soft drinks and processedfood, is proposed to be a major contributing factor to the epidemic.High fructose corn sweeteners started gaining widespread use in the foodindustry by 1967. Although glucose and fructose have the same caloricvalue per molecule, the two sugars are metabolized differently andutilize different GLUT transporters. Fructose is almost exclusivelymetabolized in the liver, and unlike the glucose metabolism pathway, thefructose metabolism pathway is not regulated by feedback inhibition bythe product (Khaitan Z et al., (2013) J. Nutr. Metab. 2013, Article ID682673, 1-12). While hexokinase and phosphofructokinase (PFK) regulatethe production of glyceraldehyde-3-P from glucose, fructokinase orketohexokinase (KHK) which is responsible for phosphorylation offructose to fructose-1-phosphate in the liver, is not down regulated byincreasing concentrations of fructose-1-phosphate. As a result, allfructose entering the cell is rapidly phosphorylated. (Cirillo P. elal., (2009) J. Am. Soc. Nephrol. 20: 545-553). Continued utilization ofATP to phosphorylate the fructose to fructose-1-phosphate results inintracellular phosphate depletion, ATP depletion, activation of AMPdeaminase and formation of uric acid (Khaitan Z. et al., (2013) J. Nutr.Metab. Article ID 682673, 1-12). Increased uric acid further stimulatesthe up-regulation of KHK (Lanaspa M. A. et al., (2012) PLOS ONE 7(10):1-11) and causes endothelial cell and adipocyte dysfunction.Fructose-1-phosphate is subsequently converted to glyceraldehyde by theaction of aldolase B and is phosphorylated toglyceraldehyde-3-phosphate. The latter proceeds downstream to theglycolysis pathway to form pyruvate, which enters the citric acid cycle,wherefrom, under well-fed conditions, citrate is exported to the cytosolfrom the mitochondria, providing Acetyl Coenzyme A for lipogenesis (FIG.1).

The phosphorylation of fructose by KHK, and subsequent activation oflipogenesis leads to, for example, fatty liver, hypertriglyceridemia,dyslipidemia, and insulin resistance. Proinflammatory changes in renalproximal tubular cells have also been shown to be induced by KHKactivity (Cirillo P. et al., (2009), J. Am. Soc. Nephrol. 20: 545-553).The phosphorylation of fructose by KHK is associated with diseases,disorders and/or conditions such as liver disease (e.g., fatty liver,steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDLcholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving. Accordingly, there is a need in the art forcompositions and methods for treating diseases, disorders, and/orconditions associated with KHK activity.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising RNAi agents,e.g., double-stranded RNAi agents, targeting ketohexokinase (KHK). Thepresent invention also provides methods of using the compositions of theinvention for inhibiting KHK expression and/or for treating a subjecthaving a disorder that would benefit from inhibiting or reducing theexpression of a KHK gene, e.g., a KHK-associated disease, such as liverdisease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving.

Accordingly, in one aspect, the present invention provides a doublestranded RNAi agent for inhibiting expression of ketohexokinase (KHK)comprising a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

In another aspect, the present inventions provides a double strandedRNAi agent for inhibiting expression of ketohexokinase (KHK), comprisinga sense strand and an antisense strand forming a double stranded region,the antisense strand comprising a region of complementarity whichcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the antisense sequences listed in any one ofTables 3, 4, and 5.

In one embodiment, the sense and antisense strands comprise sequencesare selected from the group consisting of AD-63851, AD-63820, AD-63853,AD-63839, AD-63854, AD-63855, and AD-63886 and any one of the sequencesdisclosed in any one of Tables 3, 4, 8, 11, 12, 14, and 15.

In one embodiment, the double stranded RNAi agent comprises at least onemodified nucleotide.

In one embodiment, the at least one modified nucleotide is selected fromthe group consisting of a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, a deoxy-nucleotide, a3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modifiednucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a terminal nucleotide linked to a cholesteryl derivative ora dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modifiednucleotide, a locked nucleotide, an unlocked nucleotide, aconformationally restricted nucleotide, a constrained ethyl nucleotide,an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.

In another embodiment, the at least one modified nucleotides is selectedfrom the group consisting of a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or a dodecanoic acidbisdecylamide group.

In another aspect, the present invention provides double stranded RNAiagents for inhibiting expression of ketohexokinase (KHK), which comprisea sense strand and an antisense strand forming a double stranded region,wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and the antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequences of SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:6, wherein substantially all of the nucleotides of thesense strand and substantially all of the nucleotides of the antisensestrand are modified nucleotides, and wherein the sense strand isconjugated to a ligand attached at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand are modified nucleotides.

In another embodiment, the sense strand and the antisense strandcomprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense sequences listed in any one of Tables 3, 4, 5, 6,and 7.

In some embodiments, the modified nucleotides is selected from the groupconsisting of 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, a deoxy-nucleotide, a 3′-terminaldeoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, alocked nucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitolmodified nucleotide, a cyclohexenyl modified nucleotide, a nucleotidecomprising a phosphorothioate group, a nucleotide comprising amethylphosphonate group, a nucleotide comprising a 5′-phosphate, and anucleotide comprising a 5′-phosphate mimic.

In another embodiment of the double stranded RNAi agent, at least onestrand comprises a 3′ overhang of at least 1 nucleotide. In anotherembodiment, at least one strand comprises a 3′ overhang of at least 2nucleotides.

In yet another embodiment, the double stranded RNAi agent comprises aligand. In another embodiment, the double stranded RNAi agent is aadministered using a pharmaceutical composition and a lipid formulation.In another embodiment, the lipid formulation comprises a lipidnanoparticle (LNP). In yet another embodiment, the lipid nanoparticle(LNP) comprises a MC3 lipid.

In another aspect, the present invention provides a compositioncomprising a modified antisense polynucleotide agent, wherein the agentis capable of inhibiting the expression of ketohexokinase (KHK) in acell, and comprises a sequence complementary to a sense sequenceselected from the group of the sequences listed in any one of Tables 3,4, and 5, wherein the polynucleotide is about 14 to about 30 nucleotidesin length.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agent capable of inhibiting the expression ofketohexokinase (KHK) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):

sense: 5′ n_(p)-Na-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III) 

wherein:

i, j, k, and l are each independently 0 or 1;

-   -   p, p′, q, and q′ are each independently 0-6;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may        not be present, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

-   -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; lis 1; both k and l are 0; or both k and l are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementaryto Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand.

In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13positions of the antisense strand from the 5′-end.

In one embodiment, Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

sense:  5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′ (IIIa). 

In another embodiment, formula (III) is represented by formula (IIIb):

sense:  5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′ (IIIb) 

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In yet another embodiment, formula (III) is represented by formula(IIIc):

sense:  5′ n_(p)-N_(a)-X XX-N_(b)-Y Y Y-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(a′)-n_(q′) 5′ (IIIc) 

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In a further embodiment, formula (III) is represented by formula (IIId):

sense:  5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ anti sense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′ (IIId) 

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairsin length. In another embodiment, the double-stranded region is 17-23nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 17-25 nucleotide pairs in length. In a furtherembodiment, the double-stranded region is 23-27 nucleotide pairs inlength. In another embodiment, the double-stranded region is 19-21nucleotide pairs in length. In another embodiment, the double-strandedregion is 19-23 nucleotide pairs in length. In another embodiment, thedouble-stranded region is 21-23 nucleotide pairs in length. In yetanother embodiment, each strand has 15-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro,2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment,the modifications on the nucleotides are 2′-O-methyl or 2′-fluoromodifications.

In one embodiment, the ligand the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker. Inanother embodiment, the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In another embodiment, the RNAi agent is conjugated to the ligand asshown in the following schematic

wherein X is O or S. In a specific embodiment, X is O.

In one embodiment, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In a further embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In anotherembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In anotherembodiment, the strand is the antisense strand. In a further embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In another embodiment, the strand is the antisense strand.

In another embodiment, the double stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In a further embodiment, theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus of the sense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair. In anotherembodiment, the Y nucleotides contain a 2′-fluoro modification. In afurther embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

In one embodiment, p′>0. In another embodiment, p′=2. In a furtherembodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA. In yet a further embodiment, q′=0,p=0, q=0, and p′ overhang nucleotides are non-complementary to thetarget mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In another embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage. In a further embodiment, alln_(p)′ are linked to neighboring nucleotides via phosphorothioatelinkages.

In another embodiment, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 3, 4, and 5.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of ketohexokinase (KHK). The doublestranded RNAi agents include a sense strand and an antisense strandforming a double stranded region, wherein the sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3 orSEQ ID NO:5 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, whereinsubstantially all of the nucleotides of the sense strand comprise amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoro modification wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents capable of inhibiting the expression of KHK(ketohexokinase) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(II):

sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents capable of inhibiting the expression ofketohexokinase (KHK) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):

sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In a further aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agent capable of inhibiting the expression ofketohexokinase (KHK) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):

sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3 n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein:

i, j, k, and 1 are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents capable of inhibiting the expression ofketohexokinase (KHK) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):

sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III) 

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agent capable of inhibiting the expression ofketohexokinase (KHK) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′

wherein:

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6:

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and wherein themodifications are 2′-O-methyl or 2′-fluoro modifications;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

The present invention also provides cells, vectors, host cells andpharmaceutical compositions comprising the double stranded RNAi agentsof the invention.

In one embodiment, the present invention provides double stranded RNAiagents comprising the RNAi agents listed in any one of Tables 3, 4 5, 6,and 7.

In one embodiment, a cell contains the double stranded RNAi agent.

In another embodiment, a vector encodes at least one strand of a doublestranded RNAi agent, wherein the double stranded RNAi agent comprises aregion of complementarity to at least a part of an mRNA encodingketohexokinase, wherein the double stranded RNAi agent is 30 base pairsor less in length, and wherein the double stranded RNAi agent targetsthe mRNA for cleavage. In a further embodiment, the region ofcomplementarity is at least 15 nucleotides in length. In anotherembodiment, the region of complementarity is 19 to 21 nucleotides inlength. In another embodiment, a cell contains the vector.

In some embodiments, the double stranded RNAi agent or the compositioncomprising a modified antisense polynucleotide agent is administeredusing a pharmaceutical composition.

In preferred embodiments, the double stranded RNAi agent is administeredin a solution. In some embodiments, the double stranded RNAi agent isadministered in an unbuffered solution. In another embodiment, theunbuffered solution is saline or water. In another embodiment, thedouble stranded RNAi agent is administered with a buffer solution. Inyet another embodiment, the buffer solution comprises acetate, citrate,prolamine, carbonate, or phosphate or any combination thereof. In someembodiment, the buffer solution is phosphate buffered saline (PBS).

In another aspect, the present invention provides methods of inhibitingketohexokinase (KHK) expression in a cell. The methods includecontacting the cell with the double stranded RNAi agent, apharmaceutical composition, a composition comprising a modifiedantisense polynucleotide agent, or a vector comprising the RNAi agentand maintaining the cell produced for a time sufficient to obtaindegradation of the mRNA transcript of a KHK gene, thereby inhibitingexpression of the KHK gene in the cell.

In one embodiment, the cell is within a subject. In a furtherembodiment, the subject is a human. In a further embodiment, the subjectsuffers from a ketohexokinase-associated disease.

In one embodiment, the KHK expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In another aspect, the present invention provides methods of treating asubject having a ketohexokinase (KHK)-associated disorder, comprisingadministering to the subject a therapeutically effective amount of thedouble stranded RNAi agent, a composition comprising a modifiedantisense polynucleotide agent, or a pharmaceutical compositioncomprising the double stranded RNAi agent, thereby treating the subject.

In another aspect, the present invention provides methods of treating asubject having a ketohexokinase (KHK)-associated disorder which includesubcutaneously administering to the subject a therapeutically effectiveamount of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, whereinsubstantially all of the nucleotides of the antisense strand comprise amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoromodification, wherein the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, wherein substantially all of the nucleotides of the sensestrand comprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoromodification, wherein the sensestrand comprises two phosphorothioate internucleotide linkages at the5′-terminus and, wherein the sense strand is conjugated to one or moreGalNAc derivatives attached through a branched bivalent or trivalentlinker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, the subject is a human.

In one embodiment, the ketohexokinase-associated disease is selectedfrom the group consisting liver disease, dyslipidemia, disorders ofglycemic control, cardiovascular disease, kidney disease, metabolicsyndrome, adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving. In aspecific embodiment, the liver disease is fatty liver and/orsteatohepatitis. In another embodiment, the dyslipidemia is selectedfrom the group consisting of hyperlipidemia, high LDL cholesterol, lowHDL cholesterol, hypertriglyceridemia, and postprandialhypertriglyceridemia. In yet another embodiment, the disorder ofglycemic control is insulin resistance and/or diabetes. In a furtherembodiment, the cardiovascular disease is hypertension and/orendothelial cell dysfunction. In yet another embodiment, the kidneydisease is selected from the group consisting of acute kidney disorder,tubular dysfunction, and proinflammatory changes to the proximaltubules.

In one embodiment, the double stranded RNAi agent is administered at adose of about 0.01 mg/kg to about 10 mg/kg or about 1 mg/kg to about 10mg/kg. In a preferred embodiment, the double stranded RNAi agent isadministered at a dose of about 0.1 mg/kg, about 1.0 mg/kg, or about 3.0mg/kg. In a specific embodiment, the double stranded RNAi agent isadministered at a dose of about 1 mg/kg to about 10 mg/kg.

In one embodiment, the double stranded RNAi agent is administeredsubcutaneously or intravenously.

In one embodiment, the RNAi agent is administered in two or more doses.

In yet another embodiment, the methods further comprise administering tothe subject, an additional therapeutic agent. In some embodiments, theadditional therapeutic agent is selected from the group consisting of anHMG-CoA reductase inhibitor, a diabetic therapy, an anti-hypertensivedrug, and resveratrol.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the metabolism of fructose by ketohexokinase and themetabolism of glucose and fructose by hexokinase.

FIG. 2 depicts the exon arrangement on the human KHK gene for thetranscript products of ketohexokinase A (NM_000221.2), ketohexokinase C(NM_006488.2) and transcript variant X5 (XM_005264298.1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising RNAi agents,e.g., double-stranded iRNA agents, targeting KHK. The present inventionalso provides methods of using the compositions of the invention forinhibiting KHK expression and for treating KHK-associated disease,disorders, and/or conditions, e.g., liver disease (e.g., fatty liver,steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDLcholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving (Khaitan Z. et al., (2013) J. Nutr. Metab.,Article ID 682673, 1-12; Diggle C. P. et al., (2009) J. Hisotchem.Cyochem., 57(8): 763-774; Cirillo P. et al., (2009) J. Am. Soc.Nephrol., 20: 545-553; Lanaspa M. A. et al., (2012) PLOS ONE 7(10):1-11).

The KHK (Ketohexokinase) gene is located on chromosome 2p23 and encodesketohexokinase, also known as fructokinase. KHK is a phosphotransferaseenzyme with an alcohol as the phosphate acceptor. KHK belongs to theribokinase family of carbohydrate kinases (Trinh et al., ACTA Cryst.,D65: 201-211). Two isoforms of ketohexokinase have been identified,KHK-A and KHK-C, that result from alternative splicing of the fulllength mRNA. These isoforms differ by inclusion of either exon 3a or 3c,and differ by 32 amino acids between positions 72 and 115 (see, e.g.,FIG. 2). KHK-C mRNA is expressed at high levels, predominantly in theliver, kidney and small intestine. KHK-C has a much lower K_(m) forfructose binding than KHK-A, and as a result, is highly effective inphosphorylating dietary fructose. The sequence of a human KHK-C mRNAtranscript may be found at, for example, GenBank Accession No. GI:153218447 (NM_006488.2; SEQ ID NO:1). The sequence of a human KHK-A mRNAtranscript may be found at, for example GenBank Accession No. GI:153218446 (NM_000221.2; SEQ ID NO:3). The sequence of full-length humanKHK mRNA is provided in GenBank Accession No. GI 530367552(XM_005264298.1; SEQ ID NO:5) was used (FIG. 2).

The present invention provides iRNA agents, compositions and methods formodulating the expression of a KHK gene. In certain embodiments,expression of KHK is reduced or inhibited using a KHK-specific iRNAagent, thereby leading to a decrease in the phosphorylation of fructoseto fructose-1-phosphate and thereby preventing an increase in uric acidlevels and an increase in lipogenesis. Thus, inhibition of KHK geneexpression or activity using the iRNA compositions of the invention isuseful as a therapy to reduce the lipogenic effects of dietary fructoseand preventing the concomitant accumulation of uric acid in a subject.Such inhibition is useful for treating diseases, disorders, and/orconditions such as liver disease (e.g., fatty liver, steatohepatitis),dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDLcholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia),disorders of glycemic control (e.g., insulin resistance, diabetes),cardiovascular disease (e.g., hypertension, endothelial celldyfunction), kidney disease (e.g., acute kidney disorder, tubulardysfunction, proinflammatory changes to the proximal tubules), metabolicsyndrome, adiocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving.

1. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “KHK” refers to the ketohexokinase gene or protein. KHKis also known as fructokinase. The term “KHK” includes human KHK, theamino acid and complete coding sequence of which may be found at forexample, GenBank Accession No. BC006233. The sequence of a human KHK-CmRNA transcript may be found at, for example, GenBank Accession No. GI:153218447 (NM_006488.2; SEQ ID NO:1). The reverse complement of SEQ IDNO:1 is provided as SEQ ID NO:2. The sequence of a human KHK-A mRNAtranscript may be found at, for example GenBank Accession No. GI:153218446 (NM_000221.2; SEQ ID NO:3). The reverse complement of SEQ IDNO:3 is provided as SEQ ID NO:4. The sequence of a human full-length KHKmRNA transcript is provided in GenBank Accession No. GI: 530367552(XM_005264298.1; SEQ ID NO:5). The reverse complement of SEQ ID NO:5 isprovided as SEQ ID NO:6. The sequence of mouse (Mus musculus) KHK mRNAcan be found at, for example, GenBank Accession No. GI:118130797(NM_008439.3; SEQ ID NO:7), and the reverse complement sequence isprovided at SEQ ID NO:8. The sequence of rat (Rattus rattovorus) KHKmRNA can be found at, for example GenBank Accession No. GI:126432547(NM_031855.3; SEQ ID NO:9), and the reverse complement sequence isprovided at SEQ ID NO:10. The sequence of cynomolgus monkey (Macacafascicularis) KHK mRNA, variant X1, can be found at, for example GenBankAccession No. GI:544482340 (XM_005576321.1; SEQ ID NO:11) or GI, and thereverse complement sequence is provided at SEQ ID NO:12. The sequence ofcynomolgus monkey (Macaca fascicularis) KHK mRNA, variant X3, can befound at, for example GenBank Accession No. GI:544482340(XM_005576321.1; SEQ ID NO:325) or GI, and the reverse complementsequence is provided at SEQ ID NO:326. Additional examples of KHK mRNAsequences are readily available using publicly available databases,e.g., GenBank., UniProt, OMIM, and the Macaca genome project web site

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a KHK gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a KHK gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of a KHKgene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”or “small inhibitory RNA” or “siRNA” as used interchangeably herein,refer to an agent that contains RNA as that term is defined herein, andwhich mediates the targeted cleavage of an RNA transcript via anRNA-induced silencing complex (RISC) pathway, iRNA directs thesequence-specific degradation of mRNA through a process known as RNAinterference (iRNA). The iRNA modulates, e.g., inhibits, the expressionof KHK in a cell, e.g., a cell within a subject, such as a mammaliansubject.

In one embodiment, an iRNA agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a KHKtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a KHK gene. Accordingly, the term“siRNA” is also used herein to refer to an iRNA as described above.

In another embodiment, the iRNA agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded iRNA agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima el al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded s iRNA as described herein or as chemicallymodified by the methods described in Lima el al., (2012) Cell150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a KHK gene. In some embodiments ofthe invention, a double-stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAior iRNA.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “iRNA agent” may include ribonucleotides withchemical modifications; an iRNA agent may include substantialmodifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or a modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in theagents of the invention include all types of modifications disclosedherein or known in the art. Any such modifications, as used in a siRNAtype molecule, are encompassed by “RNAi agent” for the purposes of thisspecification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected.

Where the two strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA minus any overhangs that are present in the duplex. Inaddition to the duplex structure, an iRNA may comprise one or morenucleotide overhangs.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded iRNA agent, i.e., no nucleotideoverhang. A “blunt ended” iRNA agent is a dsRNA that is double-strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The iRNA agents of the invention include iRNA agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a KHK mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., a KHK nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, 3, or 2nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding KHK). For example, a polynucleotide iscomplementary to at least a part of a KHK mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding KHK.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisenseoligonucleotide molecule that inhibits a target mRNA via an antisenseinhibition mechanism. The single-stranded antisense oligonucleotidemolecule is complementary to a sequence within the target mRNA. Thesingle-stranded antisense oligonucleotides can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The single-stranded antisense oligonucleotidemolecule may be about 15 to about 30 nucleotides in length and have asequence that is complementary to a target sequence. For example, thesingle-stranded antisense oligonucleotide molecule may comprise asequence that is at least about 15, 16, 17, 18, 19, 20, or morecontiguous nucleotides from any one of the antisense sequences describedherein.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a KHK,” as used herein, includesinhibition of expression of any KHK gene (such as, e.g., a mouse KHKgene, a rat KHK gene, a monkey KHK gene, or a human KHK gene) as well asvariants or mutants of a KHK gene that encode a KHK protein.

“Inhibiting expression of a KHK gene” includes any level of inhibitionof a KHK gene, e.g., at least partial suppression of the expression of aKHK gene, such as an inhibition by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%.

The expression of a KHK gene may be assessed based on the level of anyvariable associated with KHK gene expression, e.g., KHK mRNA level, orKHK protein level. Inhibition may be assessed by a decrease in anabsolute or relative level of one or more of these variables comparedwith a control level. The control level may be any type of control levelthat is utilized in the art, e.g., a pre-dose baseline level, or a leveldetermined from a similar subject, cell, or sample that is untreated ortreated with a control (such as, e.g., buffer only control or inactiveagent control).

In one embodiment, at least partial suppression of the expression of aKHK gene, is assessed by a reduction of the amount of KHK mRNA which canbe isolated from or detected in a first cell or group of cells in whicha KHK gene is transcribed and which has or have been treated such thatthe expression of a KHK gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition may be expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{11mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an iRNA agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an iRNA agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the iRNA agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the iRNA agent. Contacting a cell in vivo may be done, forexample, by injecting the iRNA agent into or near the tissue where thecell is located, or by injecting the iRNA agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the iRNA agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the iRNA agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an iRNA agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder and/or condition that wouldbenefit from reduction in KHK expression; a human at risk for a disease,disorder and/or condition that would benefit from reduction inketohexokinase expression; a human having a disease, disorder orcondition that would benefit from reduction in ketohexokinaseexpression.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with unwantedketohexokinase activation (e.g., liver disease (e.g., fatty liver,steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDLcholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving; stabilization (i.e., not worsening) of the oneor more diseases, disorders, and/or conditions associated withketohexokinase activity; amelioration or palliation of unwantedketohexokinase activity (e.g., phosphorylation of fructose leading toactivation of lipogenesis and increased uric acid production) whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival in the absence of treatment.

Consequently, reduction or amelioration of any of the symptoms resultingfrom fructose metabolism via KHK pathway includes reduction oramelioration of any one or more of the symptoms included but not limitedto the group comprising fatty liver, steatohepatitis, high bloodpressure, hypertension, high cholesterol, high LDL cholesterol, low HDLcholesterol, hyperlipidemia, hypertriglyceridemia, kidney disease,metabolic syndrome, excessive sugar craving, eating disorder,post-prandial hypertriglyceridemia, hepatosteatosis, gout, diabetes,acute kidney disorder, tubular dysfunction, insulin resistance andobesity. In some embodiments, the reduction or amelioration of aKHK-associated disease, disorder or condition means reduction ofhepatosteatosis, excess body fat, obesity, high cholesterol,hypertension or high blood pressure.

The term “lower” in the context of the level of ketohexokinase activityin a subject or as disease marker or symptom refers to a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or more and is preferably down toa level accepted as within the range of normal for an individual withoutsuch disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of a KHK gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., a symptom of phosphorylation offructose to fructose-1-phosphate such as activation of lipogenesis andincreased production of uric acid resulting in, for example, liverdisease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving. Thelikelihood of developing any or all of these diseases, disorders and/orconditions is reduced, for example, when an individual having one ormore risk factors for any or all of these diseases, disorders and orconditions, either fails to develop the disease, disorder and/orcondition or develops a disease, disorder and or/condition with lessseverity relative to a population having the same risk factors and notreceiving treatment as described herein. The failure to develop adisease, disorder and/or condition, or the reduction in the developmentof a symptom associated with such a disease, disorder and/or condition(e.g., by at least about 10% on a clinically accepted scale for thatdisease or disorder), or the exhibition of delayed symptoms delayed(e.g., by days, weeks, months or years) is considered effectiveprevention.

As used herein, the term “ketohexokinase associated disease” is adisease, disorder or condition that is caused by, or associated with,the ketohexokinase gene or protein, e.g., a disease, disorder orcondition caused by or associated with the phosphorylation of fructoseto fructose-1-phosphate. Such diseases are typically associated withactivation of lipogenesis and increase uric acid production.Non-limiting examples of ketohexokinase associated diseases includeliver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving.

“Therapeutically effective amount” as used herein is intended to includethe amount of an iRNA agent, that when administered to a subject havinga KHK-associated disease or disorder, is sufficient to effect treatmentof the disease or disorder (e.g., by diminishing or ameliorating thedisease or one or more symptoms of disease). The “therapeuticallyeffective amount” may vary depending on the RNAi agent, how the agent isadministered, the disease and its severity and the history, age, weight,family history, genetic makeup, the types or preceding or concomitanttreatments, if any, and other individual characteristics of the subjectto be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA agent that, when administered to a subjecthaving a KHK-associated disease but not yet (or currently) experiencingor displaying symptoms of the disease, and/or a subject at risk ofdeveloping a KHK-associated disease, e.g., a liver disease,dyslipidemia, a disorder of glycemic control, a cardiovascular disease,a kidney disease, metabolic syndrome, and/or obesity, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Ameliorating the disease includes slowing the course of thedisease or reducing the severity of later-developed disease. The“prophylactically effective amount” may vary depending on the iRNAagent, how the agent, is administered, the degree of risk of disease,and the history, age, weight, family history, genetic makeup, the typesof preceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment, iRNA agents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, urine, lymph,cerebrospinal fluid, ocular fluids, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissue(or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

The present invention also provides iRNAs which inhibit the expressionof a KHK gene. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a KHK gene in a cell, such as a cell within a subject,e.g., a mammal, such as a human having a KHK-associated disease ordisorder, including but not limited to liver disease (e.g., fatty liver,steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDLcholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving. The dsRNA includes an antisense strand having aregion of complementarity to at least a part of an mRNA formed in theexpression of a KHK gene. The region of complementarity is about 30nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contactwith a cell expressing the KHK gene, the iRNA inhibits the expression ofthe KHK gene (e.g., a human, a primate, a non-primate, or a bird KHKgene) by at least about 10% as assayed by, for example, a PCR orbranched DNA (bDNA)-based method, or by a protein-based method, such asby immunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a KHK gene.The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25,20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengthsintermediate to the above recited ranges and lengths are alsocontemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well-known in the art that dsRNAslonger than about 21-23 nucleotides in length may serve as substratesfor Dicer. As the ordinarily skilled person will also recognize, theregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to allow it to be a substrate for iRNA-directed cleavage (i.e.,cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target KHK expression is not generated in the target cell by cleavageof a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA iRNA compound can beprepared using solution-phase or solid-phase organic synthesis or both.Organic synthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables 3,4 5, 6, and 7, and the corresponding antisense strand of the sensestrand is selected from the group of sequences provided in any one ofTables 3, 4 5, 6, and 7. In this aspect, one of the two sequences iscomplementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of a KHK gene. As such, in this aspect, adsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in any one of Tables 3, 4 5, 6, and 7, andthe second oligonucleotide is described as the corresponding antisensestrand of the sense strand in any one of Tables 3, 4 5, 6, and 7. In oneembodiment, the substantially complementary sequences of the dsRNA arecontained on separate oligonucleotides. In another embodiment, thesubstantially complementary sequences of the dsRNA are contained on asingle oligonucleotide.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3, 4 5, 6, and7. dsRNAs described herein can include at least one strand of a lengthof minimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences provided in any one of Tables 3, 45, 6, and 7 minus only a few nucleotides on one or both ends can besimilarly effective as compared to the dsRNAs described above. Hence,dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or morecontiguous nucleotides derived from one of the sequences provided in anyone of Tables 3, 4 5, 6, and 7 and differing in their ability to inhibitthe expression of a KHK gene by not more than about 5, 10, 15, 20, 25,or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated to be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 3, 4 5, 6, and 7identify a site(s) in a KHK transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in any one of Tables 3, 45, 6, and 7 coupled to additional nucleotide sequences taken from theregion contiguous to the selected sequence in a KHK gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 3, 45, 6, and 7 represent effective target sequences, it is contemplatedthat further optimization of inhibition efficiency can be achieved byprogressively “walking the window” one nucleotide upstream or downstreamof the given sequences to identify sequences with equal or betterinhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 3, 4 5, 6, and 7 further optimization could beachieved by systematically either adding or removing nucleotides togenerate longer or shorter sequences and testing those sequencesgenerated by walking a window of the longer or shorter size up or downthe target RNA from that point. Again, coupling this approach togenerating new candidate targets with testing for effectiveness of iRNAsbased on those target sequences in an inhibition assay as known in theart and/or as described herein can lead to further improvements in theefficiency of inhibition. Further still, such optimized sequences can beadjusted by, e.g., the introduction of modified nucleotides as describedherein or as known in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of a KHK gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of a KHK gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of a KHK geneis important, especially if the particular region of complementarity ina KHK gene is known to have polymorphic sequence variation within thepopulation.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is unmodified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified. iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Aca, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O-N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference. Potentially stabilizing modifications to the

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded iRNA agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Provisional Application No. 61/561,710, filed onNov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, theentire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or in PCTApplication No. PCT/US2012/065691, a superior result may be obtained byintroducing one or more motifs of three identical modifications on threeconsecutive nucleotides into a sense strand and/or antisense strand ofan iRNA agent, particularly at or near the cleavage site. In someembodiments, the sense strand and antisense strand of the iRNA agent mayotherwise be completely modified. The introduction of these motifsinterrupts the modification pattern, if present, of the sense and/orantisense strand. The iRNA agent may be optionally conjugated with aGalNAc derivative ligand, for instance on the sense strand. Theresulting iRNA agents present superior gene silencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded iRNA agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an iRNA agent, the gene silencingactivity of the iRNA agent was superiorly enhanced.

Accordingly, the invention provides double-stranded iRNA agents capableof inhibiting the expression of a target gene (i.e., KHK gene) in vivo.The iRNA agent comprises a sense strand and an antisense strand. Eachstrand of the iRNA agent may range from 12-30 nucleotides in length. Forexample, each strand may be between 14-30 nucleotides in length, 17-30nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “iRNA agent” or“iRNA agent.” The duplex region of an iRNA agent may be 12-30 nucleotidepairs in length. For example, the duplex region can be between 14-30nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23nucleotide pairs in length. In another example, the duplex region isselected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27nucleotides in length.

In one embodiment, the iRNA agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the iRNAagent can each independently be a modified or unmodified nucleotideincluding, but not limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the iRNA agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The iRNA agent may contain only a single overhang, which can strengthenthe interference activity of the iRNA, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3-terminal end of the antisense strand. The iRNA may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the iRNAhas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the iRNA agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the iRNA agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the iRNA agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the iRNA agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′end, whereinone end of the iRNA agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the iRNA agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the iRNA agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the iRNA agent further comprises aligand (preferably GalNAc₃).

In one embodiment, the iRNA agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5 terminal nucleotide (position 1) positions 1to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3′ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3′ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the iRNA agent comprises sense and antisense strands,wherein the iRNA agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region which is at least 25 nucleotides in length, and thesecond strand is sufficiently complementary to a target mRNA along atleast 19 nucleotide of the second strand length to reduce target geneexpression when the iRNA agent is introduced into a mammalian cell, andwherein dicer cleavage of the iRNA agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the iRNA agentfurther comprises a ligand.

In one embodiment, the sense strand of the iRNA agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the iRNA agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an iRNA agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe iRNA from the 5′-end.

The sense strand of the iRNA agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the iRNA agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the iRNA agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the iRNA agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the iRNA agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the iRNA agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the iRNA agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the iRNA agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base: andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the iRNA agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the iRNA agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The iRNA agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand.

In one embodiment, the iRNA comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theiRNA agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In one embodiment, theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the iRNA agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mistmatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the iRNA agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the sense strand sequence may be represented byformula (I):

(I)5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the iRNA agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1′ nucleotide, from the 5′-end; oroptionally, the count starting at the 1′ paired nucleotide within theduplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

(Ib) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Id)5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the iRNA may berepresented by formula (II):

(II)5′ n_(q)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)-Y′Y′Y′-N_(b)′(X′X′X′)_(l)-N_(a)′-n_(p)′ 3′

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the iRNA agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

(IIb) 5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′N_(a)′-n_(p) 3′; (IIc)5′ n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-′X′X′X′-n_(p) 3′; or (IId)5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p) 3′.

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6. In other embodiments, k is 0 and l is 0 and the antisense strandmay be represented by the formula:

(Ia) 5′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 3′.

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the iRNA agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1′ paired nucleotidewithin the duplex region, from the 5′-end; and Y represents 2′-Fmodification. The sense strand may additionally contain XXX motif or ZZZmotifs as wing modifications at the opposite end of the duplex region;and XXX and ZZZ each independently represents a 2′-OMe modification or2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (Ha), (IIb), (IIc), and (IId),respectively.

Accordingly, the iRNA agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the iRNA duplex represented by formula (III):

(III) sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga iRNA duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb)5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIc)5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIId)5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the iRNA agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the iRNA agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the iRNA agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the iRNA agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the iRNA agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the iRNA agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the iRNA agent is represented as formula (IIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the iRNA agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the iRNA agent is represented by formula(IIId), the N, modifications are 2′-O-methyl or 2′-fluoro modificationsand n_(p)′>0 and at least one n_(p)′ is linked to a neighboringnucleotide a via phosphorothioate linkage. In yet another embodiment,when the iRNA agent is represented by formula (IIId), the N_(a)modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 andat least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the iRNAagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the iRNA agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the iRNA agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the iRNA agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two iRNA agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric iRNA agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the iRNA agent that containsconjugations of one or more carbohydrate moieties to a iRNA agent canoptimize one or more properties of the iRNA agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the iRNAagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The iRNA agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the iRNA agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 3, 4 5, 6, and 7. These agents may further comprise aligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 14) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 15) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 16)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the presentinvention include those described in PCT Publication Nos. WO 2014/179620and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(RN), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) (represent the ligand; i.e. each independently for eachoccurrence a monosaccharide (such as GalNAc), disaccharide,trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andR^(a) is H or amino acid side chain. Trivalent conjugating GalNAcderivatives are particularly useful for use with RNAi agents forinhibiting the expression of a target gene, such as those of formula(XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. (hem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO.J., 1991, 10:111; Kabanov et al., FEBS Left., 1990, 259:327; Svinarchuket al., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disease, disorder or condition associated withphosphorylation of fructose by KHK) can be achieved in a number ofdifferent ways. For example, delivery may be performed by contacting acell with an iRNA of the invention either in vitro or in vivo. In vivodelivery may also be performed directly by administering a compositioncomprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivodelivery may be performed indirectly by administering one or morevectors that encode and direct the expression of the iRNA. Thesealternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003)Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004)J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects, iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., eta/(2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et a(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the C5 gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty el al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a KHK gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery, e.g., bysubcutaneous (SC) or intravenous (IV) delivery. Another example iscompositions that are formulated for direct delivery into the brainparenchyma, e.g., by infusion into the brain, such as by continuous pumpinfusion. The pharmaceutical compositions of the invention may beadministered in dosages sufficient to inhibit expression of a KHK gene.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A, iRNA formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate iRNA. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987. U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. A. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, el al.Endocrinol. 115.757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(MI), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(MI), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(MI) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNA agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad Sci.USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA agentcan be delivered, for example, subcutaneously by infection in order todeliver iRNA agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0f 20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml,Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,31-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~1:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN-100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxyclodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP20 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference. XTC comprising formulations are described, e.g., in U.S.Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. ProvisionalSer. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filedJun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24,2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference. MC3 comprising formulationsare described, e.g., in U.S. Publication No. 2010/0324120, filed Jun.10, 2010, the entire contents of which are hereby incorporated byreference. ALNY-100 comprising formulations are described, e.g,.International patent application number PCT/US09/63933, filed on Nov.10, 2009, which is hereby incorporated by reference. C12-200 comprisingformulations are described in U.S. Provisional Ser. No. 61/175,770,filed May 5, 2009 and International Application No. PCT/US10/33777,filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —S OnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an. Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield:−6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199: Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245, Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories-surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enaminesxsee e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen: Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega:Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass' D1Transfection Reagent (New England Biolabs: Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International: Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-iRNA mechanism and which are useful intreating a hemolytic disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby KHK expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VII. Methods of the Invention

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having, or prone to developing,a KHK-associated disease, disorder, and/or condition (e.g., liverdisease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving aniRNA agent, pharmaceutical compositions comprising an iRNA agent, orvector comprising an iRNA of the invention.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in KHKexpression, e.g., a KHK-associated disease, e.g., liver disease (e.g.,fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, highLDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving. The treatment methods (and uses) of theinvention include administering to the subject, e.g., a human, atherapeutically effective amount of an iRNA agent targeting a KHK geneor a pharmaceutical composition comprising an iRNA agent targeting a KHKgene, thereby treating the subject having a disorder that would benefitfrom reduction in KHK expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin KHK expression, e.g., a KHK-associated disease, e.g., liver disease(e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving. Themethods include administering to the subject a therapeutically effectiveamount of the iRNA agent, e.g., dsRNA, or vector of the invention,thereby preventing at least one symptom in the subject having a disorderthat would benefit from reduction in KHK expression. For example, theinvention provides methods for preventing lipogenesis and/orhyperuricemia in a subject suffering from a disorder that would benefitfrom reduction in KHK expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of KHK expression.

In a further aspect, the present invention provides uses of an iRNAagent, e.g., a dsRNA, of the invention targeting a KHK gene orpharmaceutical composition comprising an iRNA agent targeting a KHK genein the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of KHKexpression, such as a subject having a disorder that would benefit fromreduction in KHK expression, e.g., a KHK-associated disease, e.g., liverdisease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of KHK expression, such as a KHK-associated disease, e.g.,liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g.,hyperlipidemia, high LDL cholesterol, low HDL cholesterol,hypertriglyceridemia, postprandial hypertriglyceridemia), disorders ofglycemic control (e.g., insulin resistance, diabetes), cardiovasculardisease (e.g., hypertension, endothelial cell dysfunction), kidneydisease (e.g., acute kidney disorder, tubular dysfunction,proinflammatory changes to the proximal tubules), metabolic syndrome,adipocyte dysfunction, visceral adipose deposition, obesity,hyperuricemia, gout, eating disorders, and excessive sugar craving.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of KHK expression, such as aKHK-associated disease, e.g., liver disease (e.g., fatty liver,steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDLcholesterol, low HDL cholesterol, hypertriglyceridemia, postprandialhypertriglyceridemia), disorders of glycemic control (e.g., insulinresistance, diabetes), cardiovascular disease (e.g., hypertension,endothelial cell dysfunction), kidney disease (e.g., acute kidneydisorder, tubular dysfunction, proinflammatory changes to the proximaltubules), metabolic syndrome, adipocyte dysfunction, visceral adiposedeposition, obesity, hyperuricemia, gout, eating disorders, andexcessive sugar craving.

In one embodiment, an iRNA agent targeting KHK is administered to asubject having a KHK-associated disease such that KHK levels, e.g., in acell, tissue, blood or other tissue or fluid of the subject are reducedby at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or morewhen the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering acomposition described herein such that expression of the target KHK geneis decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours.In one embodiment, expression of the target KHK gene is decreased for anextended duration, e.g., at least about two, three, four, five, six,seven days or more, e.g., about one week, two weeks, three weeks, orabout four weeks or longer.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with aKHK-associated disease. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of dyslipidemia may be assessed, forexample, by periodic monitoring of LDL cholesterol, HDL cholesterol andtriglyceride levels. In a further example, efficacy of treatment of aglucose control disorder may be assessed, for example, by periodicmonitoring of insulin and glucose levels. In another example, efficacyof treatment of obesity may be assessed, for example by periodicmonitoring of body mass index. In yet another example, efficacy oftreatment of hypertension may be assessed, for example, by periodicmonitoring of blood pressure. Comparison of the later readings with theinitial readings provide a physician an indication of whether thetreatment is effective. It is well within the ability of one skilled inthe art to monitor efficacy of treatment or prevention by measuring anyone of such parameters, or any combination of parameters. In connectionwith the administration of an iRNA targeting KHK or pharmaceuticalcomposition thereof, “effective against” a KHK-associated diseaseindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating a KHK-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as weekly,biweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration weekly or biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.

Administration of the iRNA can reduce KHK levels, e.g., in a cell,tissue, blood, urine or other compartment of the patient by at leastabout 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion, and monitored foradverse effects, such as an allergic reaction. In another example, thepatient can be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on KHK expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where themodified or unmodified iRNA agent is directly suspended in aqueous orsuitable buffer solvent, as a “free iRNA.” A free iRNA is administeredin the absence of a pharmaceutical composition. The free iRNA may be ina suitable buffer solution. The buffer solution may comprise acetate,citrate, prolamine, carbonate, or phosphate, or any combination thereof.In one embodiment, the buffer solution is phosphate buffered saline(PBS). The pH and osmolarity of the buffer solution containing the iRNAcan be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of KHKgene expression are those having a KHK-associated disease or disorder asdescribed herein. In one embodiment, a subject having a KHK-associateddisease has liver disease (e.g., fatty liver, steatohepatitis). Inanother embodiment, a subject having a KHK-associate disease hasdyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDLcholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia).In another embodiment, a subject having a KHK-associate disease has adisorder of glycemic control (e.g., insulin resistance, diabetes). Inyet another embodiment, a subject having a KHK-associate disease hascardiovascular disease (e.g., hypertension, endothelial celldysfunction). In one embodiment, a subject having a KHK-associatedisease has kidney disease (e.g., acute kidney disorder, tubulardysfunction, proinflammatory changes to the proximal tubules). Inanother embodiment, a subject having a KHK-associate disease hasmetabolic syndrome. In a particular embodiment, a subject having aKHK-associate disease has adipocyte dysfunction. In yet anotherembodiment, a subject having a KHK-associate disease has visceraladipose deposition. In another embodiment, a subject having aKHK-associate disease has obesity. In a particular embodiment, a subjecthaving a KHK-associate disease has hyperuricemia. In another embodiment,a subject having a KHK-associate disease has gout. In anotherembodiment, a subject having a KHK-associate disease has an eatingdisorder and/or excessive sugar craving.

Treatment of a subject that would benefit from a reduction and/orinhibition of KHK gene expression includes therapeutic and prophylactictreatment.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof for treating a subject that wouldbenefit from reduction and/or inhibition of KHK expression, e.g., asubject having a KHK-associated disease, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, in certain embodiments, an iRNA targeting KHK is administeredin combination with, e.g., an agent useful in treating a KHK-associateddisease as described elsewhere herein. For example, additionaltherapeutics and therapeutic methods suitable for treating a subjectthat would benefit from reduction in KHK expression, e.g., a subjecthaving a KHK-associated disease, include an HMG-CoA reductase inhibitor,a diabetic therapy, an anti-hypertensive drug, resveratrol, or othertherapeutic agents for treating a KHK-associated disease. ExemplaryHMG-CoA reductase inhibitors include atorvastatin (Pfizer'sLipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-MyersSquibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck'sZocorg/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas),lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa;Schwarz Pharma's Liposcler), fluvastatin (Novartis'Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin(Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca'sCrestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical,Kowa Kogyo, Sankyo, and Novartis). Exemplary diabetic therapies areknown in the art and include, for example, insulin sensitizers, such asbiguanides (e.g., metformin) and thiazolidinediones (e.g.,rosiglitazone, pioglitazone, troglitazone); secretagogues, such as thesulfonylureas (e.g., glyburide, glipizide, glimepiride, tolbutamide,acetohexamide, tolazamide, chlorpropamide, gliclazide, glycopyamide,gliquidone), the nonsulfonylurea secretagogues, e.g., meglitinidederivatives (e.g., repaglinide, nateglinide); the dipeptidyl peptidaseIV inhibitors (e.g., sitagliptin, saxagliptin, linagliptin,vildagliptin, allogliptin, septagliptin): alpha-glucosidase inhibitors(e.g., acarbose, miglitol, voglibose); amylinomimetics (e.g.,pramlintide acetate); incretin mimetics (e.g., exenatide, liraglutide,taspoglutide); insulin and its analogues (e.g., rapid acting, slowacting, and intermediate acting); bile acid sequestrants (e.g.,colesevelam); and dopamine agonists (e.g., bromocriptine), alone or incombinations. Exemplary anti-hypertensive drugs are known in the art andinclude diuretics (e.g., thiazide diuretics (e.g., chlorothiazide,chlorthalidone, hydrochlorothiazide, indapamide, metolazone), loopdiuretics (e.g., bumetanide, ethacrynic acid, furosemide, torsemide),and potassium-sparing diuretics/aldosterone-receptor blockers (e.g.,amiloride, spironolactone, triamterene, eplerenone)), anti-adrenergicdrugs (e.g., beta blockers (e.g., atenolol, metoprolol, metoprololextended release, nebivolol, nadolol, pindolol, propranolol, sotalol,timolol), alpha-1-blockers (e.g., doxazosin, prazosin, terazosin), alphaand beta blockers (e.g., carvedilol, labetalol), centrally acting agents(e.g., clonidine, methyldopa), peripheral nerve-acting agents (e.g.,guanethidine, reserpine), and direct-acting vasodilators (e.g.,hydralazine, minoxidil)), calcium channel blockers (e.g., amlodipine,diltiazem, felodipine, isradipine, nicardipine, nifedipine, verapamil),ace inhibitors (e.g., benazepril, captopril, enalapril, fosinopril,lisinopril, quinapril, ramipril), angiotensin-receptor blockers (e.g.,candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan,valsartan) or any combinations thereof.

The iRNA agent and an additional therapeutic agent and/or treatment maybe administered at the same time and/or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times and/or by anothermethod known in the art or described herein.

The present invention also provides methods of using an iRNA agent ofthe invention and/or a composition containing an iRNA agent of theinvention to reduce and/or inhibit KHK expression in a cell. In otheraspects, the present invention provides an iRNA of the invention and/ora composition comprising an iRNA of the invention for use in reducingand/or inhibiting KHK expression in a cell. In yet other aspects, use ofan iRNA of the invention and/or a composition comprising an iRNA of theinvention for the manufacture of a medicament for reducing and/orinhibiting KHK expression in a cell are provided.

The methods and uses include contacting the cell with an iRNA, e.g., adsRNA, of the invention and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of a KHK gene, therebyinhibiting expression of the KHK gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of KHK may be determinedby determining the mRNA expression level of KHK using methods routine toone of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, bydetermining the protein level of KHK using methods routine to one ofordinary skill in the art, such as Western blotting, immunologicaltechniques, flow cytometry methods, ELISA, and/or by determining abiological activity of KHK (e.g., phosphorylation of fructose tofructose-1-phosphate). In one embodiment, reduction in KHK geneexpression can be determined by measuring the level of fructose in theurine.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses a KHK gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

KHK expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the KHK gene of the mammal to be treated. When theorganism to be treated is a human, the composition can be administeredby any means known in the art including, but not limited tosubcutaneous, intravenous, oral, intraperitoneal, or parenteral routes,including intracranial (e.g., intraventricular, intraparenchymal andintrathecal), intramuscular, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by subcutaneousor intravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof KHK, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of a KHK gene in a mammal, e.g., a human. Thepresent invention also provides a composition comprising an iRNA, e.g.,a dsRNA, that targets a KHK gene in a cell of a mammal for use ininhibiting expression of the KHK gene in the mammal. In another aspect,the present invention provides use of an iRNA, e.g., a dsRNA, thattargets a KHK gene in a cell of a mammal in the manufacture of amedicament for inhibiting expression of the KHK gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an iRNA, e.g., a dsRNA, that targets a KHK genein a cell of the mammal and maintaining the mammal for a time sufficientto obtain degradation of the mRNA transcript of the KHK gene, therebyinhibiting expression of the KHK gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sampleof the iRNA-administered subject by any methods known it the art, e.g.qRT-PCR, described herein. Reduction in protein production can beassessed by any methods known it the art and by methods, e.g., ELISA orWestern blotting, described herein. In one embodiment, a puncture liverbiopsy sample serves as the tissue material for monitoring the reductionin KHK gene and/or protein expression. In another embodiment, a bloodsample serves as the tissue material for monitoring the reduction in KHKgene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target invivo following administration of iRNA agent is done by performing5′-RACE or modifications of the protocol as known in the art (Lasham Aet al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al.(2006) Nature 441: 111-4).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1, iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

A detailed list of KHK sense and antisense strand sequences is shown inTables 3, 4 and 5.

Transcripts

siRNA design was carried out to identify siRNAs targeting human,cynomolgus monkey (Macaca fascicularis; henceforth “cyno”), mouse, andrat KHK transcripts annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/). Design used the followingtranscripts from the NCBI RefSeq collection: Human—XM_005264298.1;Cyno—XM_005576324.1; Mouse—NM_008439.3; Rat—NM_031855.3. Due to highprimate/rodent sequence divergence, siRNA duplexes were designed inseveral separate batches, including but not limited to batchescontaining duplexes matching human and cyno transcripts only; human,cyno, and mouse transcripts only; and human, cyno, mouse, and rattranscripts only. Most siRNA duplexes were designed that shared 100%identity in the designated region with the listed human transcript andother species transcripts considered in each design batch (above). Insome instances, mismatches between duplex and mRNA target were allowedat the first antisense (last sense) position when the antisensestrand:target mRNA complementary basepair was a GC or CG pair. In thesecases, duplexes were designed with UA or AU pairs at the firstantisense:last sense pair. Thus the duplexes maintained complementaritybut were mismatched with respect to target (U:C, U:G, A:C, or A:G).

siRNA Design, Specificity, and Efficacy Prediction

The specificity of all possible 19mers was predicted from each sequence.Candidate 19mers that lacked repeats longer than 7 nucleotides were thenselected. These 476 candidate human/cyno, 71 human/cyno/mouse, and 58human/cyno/mouse/rat siRNAs were used in comprehensive searches againstthe appropriate transcriptomes (defined as the set of NM_ and XM_recordswithin the human, cyno, mouse, or rat NCBI Refseq sets) using anexhaustive “brute-force” algorithm implemented in the python script‘BruteForce.py’. The script next parsed the transcript-oligo alignmentsto generate a score based on the position and number of mismatchesbetween the siRNA and any potential ‘off-target’ transcript. Theoff-target score is weighted to emphasize differences in the ‘seed’region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Eacholigo-transcript pair from the brute-force search was given a mismatchscore by summing the individual mismatch scores; mismatches in theposition 2-9 were counted as 2.8, mismatches in the cleavage sitepositions 10-11 were counted as 1.2, and mismatches in region 12-19counted as 1.0. An additional off-target prediction was carried out bycomparing the frequency of heptamers and octomers derived from 3distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start were used to create 2 heptamersand one octomer. Heptamer1 was created by adding a 3′ A to the hexamer;heptamer2 was created by adding a 5′ A to the hexamer; the octomer wascreated by adding an A to both 5′ and 3′ ends of the hexamer. Thefrequency of octomers and heptamers in the human, cyno, mouse, or rat3′UTRome (defined as the subsequence of the transcriptome from NCBI'sRefseq database where the end of the coding region, the ‘CDS’, isclearly defined) was pre-calculated. The octomer frequency wasnormalized to the heptamer frequency using the median value from therange of octomer frequencies. A ‘mirSeedScore’ was then calculated bycalculating the sum of ((3×normalized octomer count)+(2×heptamer2count)+(1×heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2 and 2.8 as moderately specific.siRNA strands were sorted by the specificity of the antisense strand.Moderately (or higher) specific duplexes whose antisense oligospossessed characteristics of duplexes with high predicted efficacy,including maximal UA content in the seed region and low overall GCcontent were selected. One additional duplex with an antisense score of1.2 in the rat (but >=2 in the other species) was also included.

Candidate GalNaC-conjugated duplexes, 21 and 23 nucleotides long on thesense and antisense strands respectively, were designed by extendingantisense 19mers (described above) to 23 nucleotides oftarget-complementary sequence. All species transcripts included in thedesign batch were checked for complementarity. For each duplex, thesense 21mer was specified as the reverse complement of the first 21nucleotides of the antisense strand.

siRNA Sequence Selection

A total of 21 sense and 21 antisense derived human/cyno/mouse/rat siRNA21/23mer oligos (Table 3), 29 sense and 29 antisense derived human/cynosiRNA 21/23mer oligos (Table 4) and 3 sense and 3 antisense derivedhuman/cyno/mouse (Table 5) siRNA 21/23mer oligos were synthesized.

siRNA Synthesis

General Small and Medium Scale RNA Synthesis Procedure

RNA oligonucleotides are synthesized at scales between 0.2-500 μmolusing commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and2-N-isobutyrylguanosine and the corresponding 2′-O-methyl and 2′-fluorophosphoramidites according to standard solid phase oligonucleotidesynthesis protocols. The amidite solutions are prepared at 0.1-0.15 Mconcentration and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile)is used as the activator. Phosphorothioate backbone modifications areintroduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) inlutidine:acetonitrile (1:1) (v; v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidationstep. After completion of synthesis, the sequences are cleaved from thesolid support and deprotected using methylamine followed bytriethylamine.3HF to remove any 2′-O-t-butyldimethylsilyl protectinggroups present.

For synthesis scales between 5-500 μmol and fully 2′ modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides are deprotected using 3:1 (v/v) ethanol andconcentrated (28-32%) aqueous ammonia either at 35° C. 16 h or 55° C.for 5.5 h. Prior to ammonia deprotection the oligonucleotides aretreated with 0.5 M piperidine in acetonitrile for 20 min on the solidsupport. The crude oligonucleotides are analyzed by LC-MS andanion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides iscarried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5(buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH=8.5 (bufferB). Fractions are analyzed for purity by analytical HPLC. Theproduct-containing fractions with suitable purity are pooled andconcentrated on a rotary evaporator prior to desalting. The samples aredesalted by size exclusion chromatography and lyophilized to dryness.Equal molar amounts of sense and antisense strands are annealed in 1×PBSbuffer to prepare the corresponding siRNA duplexes.

For small scales (0.2-1 μmol), synthesis is performed on a MerMade 192synthesizer in a 96 well format. In case of fully 2′-modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides are deprotected using methylamine at room temperaturefor 30-60 min followed by incubation at 60° C. for 30 min or using 3:1(v/v) ethanol and concentrated (28-32%) aqueous ammonia at roomtemperature for 30-60 min followed by incubation at 40° C. for 1.5hours. The crude oligonucleotides are then precipitated in a solution ofacetonitrile:acetone (9:1) and isolated by centrifugation and decantingthe supernatant. The crude oligonucleotide pellet is re-suspended in 20mM NaOAc buffer and analyzed by LC-MS and anion exchange HPLC. The crudeoligonucleotide sequences are desalted in 96 deep well plates on a 5 mLHiTrap Sephadex G25 column (GE Healthcare). In each well about 1.5 mLsamples corresponding to an individual sequence is collected. Thesepurified desalted oligonucleotides are analyzed by LC-MS and anionexchange chromatography. Duplexes are prepared by annealing equimolaramounts of sense and antisense sequences on a Tecan robot. Concentrationof duplexes is adjusted to 10 μM in 1×PBS buffer.

Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo Analysis

Oligonucleotides conjugated with GalNAc ligand at their 3′-terminus aresynthesized at scales between 0.2-500 μmol using a solid supportpre-loaded with a Y-shaped linker bearing a 4,4′-dimethoxytrityl(DMT)-protected primary hydroxy group for oligonucleotide synthesis anda GalNAc ligand attached through a tether.

For synthesis of GalNAc conjugates in the scales between 5-500 μmol, theabove synthesis protocol for RNA is followed with the followingadaptions: For polystyrene-based synthesis supports 5% dichloroaceticacid in toluene is used for DMT-cleavage during synthesis. Cleavage fromthe support and deprotection is performed as described above.Phosphorothioate-rich sequences (usually >5 phorphorothioates) aresynthesized without removing the final 5′-DMT group (“DMT-on”) and,after cleavage and deprotection as described above, purified by reversephase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mMammoniumacetate in 80% acetonitirile (buffer B). Fractions are analyzedfor purity by analytical HPLC and/or LC-MS. The product-containingfractions with suitable purity are pooled and concentrated on a rotaryevaporator. The DMT-group is removed using 20%-25% acetic acid in wateruntil completion. The samples are desalted by size exclusionchromatography and lyophilized to dryness. Equal molar amounts of senseand antisense strands are annealed in 1×PBS buffer to prepare thecorresponding siRNA duplexes.

For small scale synthesis of GalNAc conjugates (0.2-1 μmol), includingsequences with multiple phosphorothioate linkages, the protocolsdescribed above for synthesis of RNA or fully 2′-F/2′-OMe-containingsequences on MerMade platform are applied. Synthesis is performed onpre-packed columns containing GalNAc-functionalized controlled poreglass support.

Example 2. General In Vitro Screening of siRNA Duplexes

The in vitro efficacy of the duplexes can be determined in single dosescreens for any RNAi targeted gene expression using the followingmethods. Similar methods may be used for multi-dose screens to determinethe dose response of the duplexes and to calculate the IC₅₀ of theduplexes.

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) are grown to near confluence at 37° C.in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Cells are washed andre-suspended at 0.25×10⁶ cells/ml. During transfections, cells areplated onto a 96-well plate with about 20,000 cells per well.

Primary mouse hepatocytes (PMH) are freshly isolated from a C57BL/6female mouse (Charles River Labortories International, Inc. Willmington,Mass.) less than 1 hour prior to transfections and grown in primaryhepatocyte media. Cells are resuspended at 0.11×106 cells/ml inInVitroGRO CP Rat (plating) medium (Celsis In Vitro Technologies,catalog number SO1494). During transfections, cells are plated onto a BDBioCoat 96 well collagen plate (BD, 356407) at 10,000 cells per well andincubated at 37° C. in an atmosphere of 5% CO2.

Cryopreserved Primary Cynomolgus Hepatocytes (Celsis In VitroTechnologies, M003055-P) are thawed at 37° C. water bath immediatelyprior to usage and re-suspended at 0.26×10⁶ cells/ml in InVitroGRO CP(plating) medium (Celsis In Vitro Technologies, catalog number Z99029).During transfections, cells are plated onto a BD BioCoat 96 wellcollagen plate (BD, 356407) at 25,000 cells per well and incubated at37° C. in an atmosphere of 5% CO2.

For Hep3B, PMH, and primary Cynomolgus hepatocytes, transfection arecarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. catalog number 13778-150)to 5 μl of each siRNA duplex to an individual well in a 96-well plate.The mixture is then incubated at room temperature for 20 minutes. Eightyμl of complete growth media without antibiotic containing theappropriate cell number are then added to the siRNA mixture. Cells areincubated for 24 hours prior to RNA purification.

Single dose experiments are performed at 10 nM and 0.1 nM final duplexconcentration for GalNAc modified sequences or at 1 nM and 0.01 nM finalduplex concentration for all other sequences. Dose response experimentsare done at 3, 1, 0.3, 0.1, 0.037, 0.0123, 0.00412, and 0.00137 nM finalduplex concentration for primary mouse hepatocytes and at 3, 1, 0.3,0.1, 0.037, 0.0123, 0.00412, 0.00137, 0.00046, 0.00015, 0.00005, and0.000017 nM final duplex concentration for Hep3B cells.

Free Uptake Transfection

Free uptake experiments are performed by adding 10 μl of siRNA duplexesin PBS per well into a 96 well plate. Ninety μl of complete growth mediacontaining appropriate cell number for the cell type is then added tothe siRNA. Cells are incubated for 24 hours prior to RNA purification.Single dose experiments are performed at 500 nM and 5 nM final duplexconcentration and dose response experiments are done at 1000, 333, 111,37, 12.3, 4.12, 1.37, 0.46 nM final duplex concentration.

Total RNA Isolation Using DYNABEAADS mRNA Isolation Kit (Invitrogen,Part #: 610-12)

Cells are harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed is the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture are added to around bottom plate and mixed for 1 minute. Magnetic beads are capturedusing a magnetic stand and the supernatant is removed without disturbingthe beads. After removing the supernatant, the lysed cells are added tothe remaining beads and mixed for 5 minutes. After removing thesupernatant, magnetic beads are washed 2 times with 150 μl Wash Buffer Aand mixed for 1 minute. The beads are captured again and the supernatantis removed. The beads are then washed with 150 μl Wash Buffer B,captured and the supernatant is removed. The beads are next washed with150 μl Elution Buffer, captured and the supernatant removed. Finally,the beads are allowed to dry for 2 minutes. After drying, 50 μl ofElution Buffer is added and mixed for 5 minutes at 70° C. The beads arecaptured on a magnet for 5 minutes. Forty-five μl of supernatant isremoved and added to another 96 well plate.

General cDNA Synthesis Using ABI High Capacity cDNA ReverseTranscription Kit (Applied Biosystems, Foster City, Calif., Cat#4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction is prepared. Equal volumes master mix and RNA are mixed for afinal volume of 12 μl for in vitro screened or 20 μl for in vivoscreened samples. cDNA is generated using a Bio-Rad C-1000 or S-1000thermal cycler (Hercules, Calif.) through the following steps: 25° C.for 10 minutes, 37° C. for 120 minutes, 85° C. for 5 seconds, and 4° C.hold.

Real Time PCR

Two μl of cDNA are added to a master mix containing 2 μl of H₂O, 0.5 μlGAPDH TaqMan Probe (Life Technologies catalog number 4326317E for Hep3Bcells, catalog number 352339E for primary mouse hepatocytes or customprobe for cynomolgus primary hepatocytes), 0.5 μl C5 TaqMan probe (LifeTechnologies c catalog number Hs00156197_m1 for Hep3B cells ormm00439275_m1 for Primary Mouse Hepatoctyes or custom probe forcynomolgus primary hepatocytes) and 5 μl Lightcycler 480 probe mastermix (Roche catalog number 04887301001) per well in a 384 well plates(Roche catalog number 04887301001). Real time PCR is performed in anRoche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Forin vitro screening, each duplex is tested with two biological replicatesunless otherwise noted and each Real Time PCR is performed in duplicatetechnical replicates. For in vivo screening, each duplex is tested inone or more experiments (3 mice per group) and each Real Time PCR is runin duplicate technical replicates.

To calculate relative fold change in KHK mRNA levels, real time data areanalyzed using the ΔΔCt method and normalized to assays performed withcells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀sare calculated using a 4 parameter fit model using XLFit and normalizedto cells transfected with AD-1955 over the same dose range, or to itsown lowest dose.

The sense and antisense sequences of AD-1955 are:

(SEQ ID NO: 17) SENSE: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 18)ANTISENSE: UCGAAGuACUcAGCGuAAGdTsdT

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nueleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-triethyluridine-3′-phosphate Tf2′-fluoro-5-methylutidine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinolHyp-(GalNAc-alkyl)3

TABLE 3 Unmodified Sense and Antisense Strand Sequences of KHK dsRNAsRef Seq; Corresponding mRNA Target SEQ SEQ position of sense strand SiteSense (5′-3′) ID NO: Antisense (5′-3′) ID NO: XM_005264298_816-838_s 816UGUCAGCAAAGAUGUGGCCAA 19 UUGGCCACAUCUUUGCUGACAAA 40XM_005264298_504-526_s 504 AGAGAAGCAGAUCCUGUGCGU 20ACGCACAGGAUCUGCUUCUCUUC 41 XM_005264298_810-832_s 810GGUGUUUGUCAGCAAAGAUGU 21 ACAUCUUUGCUGACAAACACCAC 42XM_005264298_651-673_s 651 GAUCCACAUUGAGGGCCGGAA 22UUCCGGCCCUCAAUGUGGAUCCA 43 XM_005264298_815_837_s 815UUGUCAGCAAAGAUGUGGCCA 23 UGGCCACAUCUUUGCUGACAAAC 44XM_005264298_650-672_s 650 GGAUCCACAUUGAGGGCCGGA 24UCCGGCCCUCAAUGUGGAUCCAC 45 XM_005264298_510_532_s 510GCAGAUCCUGUGCGUGGGGCU 25 AGCCCCACGCACAGGAUCUGCUU 46XM_005264298_813_835_C21A_s 813 GUUUGUCAGCAAAGAUGUGGA 26UCCACAUCUUUGCUGACAAACAC 47 XM_005264298_505-527_G21A_s 505GAGAAGCAGAUCCUGUGCGUA 27 UACGCACAGGAUCUGCUUCUCUU 48XM_005264298_644-666-G21A_s 644 UCAAGUGGAUCCACAUUGAGA 28UCUCAAUGUGGAUCCACUUGAAC 49 XM_005264298_648-670_G21A_s 648GUGGAUCCACAUUGAGGGCCA 29 UGGCCCUCAAUGUGGAUCCACUU 50XM_005264298_646-668_C21A_s 646 AAGUGGAUCCACAUUGAGGGA 30UCCCUCAAUGUGGAUCCACUUGA 51 XM_005264298_811-833_G21A_s 811GUGUUUGUCAGCAAAGAUGUA 31 UACAUCUUUGCUGACAAACACCA 52XM_005264298_812-834_G21A_s 812 UGUUUGUCAGCAAAGAUGUGA 32UCACAUCUUUGCUGACAAACACC 53 XM_005264298_649-671_G21A_s 649UGGAUCCACAUUGAGGGCCGA 33 UCGGCCCUCAAUGUGGAUCCACU 54XM_005264298_507-529_G21A_s 507 GAAGCAGAUCCUGUGCGUGGA 34UCCACGCACAGGAUCUGCUUCUC 55 XM_005264298_502-524_C21A_s 502GAAGAGAAGCAGAUCCUGUGA 35 UCACAGGAUCUGCUUCUCUUCCA 56XM_005264298_645-667_G21A_s 645 CAAGUGGAUCCACAUUGAGGA 36UCCUCAAUGUGGAUCCACUUGAA 57 XM_005264298_647-669_C21A_s 647AGUGGAUCCACAUUGAGGGCA 37 UGCCCUCAAUGUGGAUCCACUUG 58XM_005264298_503-525_G21A_s 503 AAGAGAAGCAGAUCCUGUGCA 38UGCACAGGAUCUGCUUCUCUUCC 59 XM_005264298_506-528_G21A_s 506AGAAGCAGAUCCUGUGCGUGA 39 UCACGCACAGGAUCUGCUUCUCU 60

TABLE 4 Unmodified Sense and Antisense Strand Sequences of KHK dsRNAsSEQ SEQ Ref Seq; Corresponding mRNA Target ID IDposition of sense strand Site Sense (5′-3′) NO: Antisense (5′-3′) NO:XM_005264298.1_593-615_s 593 GCCUGCCAGAUGUGUCUGCUA 61UAGCAGACACAUCUGGCAGGCUC  90 XM_005264298.1_642-664_s 642GUUCAAGUGGAUCCACAUUGA 62 UCAAUGUGGAUCCACUUGAACUG  91XM_005264298.1_640-662_s 640 CAGUUCAAGUGGAUCCACAUU 63AAUGUGGAUCCACUUGAACUGGG  92 XM_005264298.1_808-830_s 808GUGGUGUUUGUCAGCAAAGAU 64 AUCUUUGCUGACAAACACCACGU  93XM_005264298.1_643-665_G21A_s 643 UUCAAGUGGAUCCACAUUGAA 65UUCAAUGUGGAUCCACUUGAACU  94 XM_005264298.1_806-828_G21U_s 806ACGUGGUGUUUGUCAGCAAAU 66 AUUUGCUGACAAACACCACGUCU  95XM_005264298.1_641-663_G21A_s 641 AGUUCAAGUGGAUCCACAUUA 67UAAUGUGGAUCCACUUGAACUGG  96 XM_005264298.1_877-899_s 877UUGUAUGGUCGUGUGAGGAAA 68 UUUCCUCACACGACCAUACAAGC  97XM_005264298.1_795-817_s 795 UGGCUACGGAGACGUGGUGUU 69AACACCACGUCUCCGUAGCCAAA  98 XM_005264298.1_828-850_s 828UGUGGCCAAGCACUUGGGGUU 70 AACCCCAAGUGCUUGGCCACAUC  99XM_005264298.1_639-661_s 639 CCAGUUCAAGUGGAUCCACAU 71AUGUGGAUCCACUUGAACUGGGU 100 XM_005264298.1_804-826_s 804AGACGUGGUGUUUGUCAGCAA 72 UUGCUGACAAACACCACGUCUCC 101XM_005264298.1_555-577_s 555 GGUGGACAAGUACCCUAAGGA 73UCCUUAGGGUACUUGUCCACCAG 102 XM_005264298.1_632-654_s 632AUCUGACCCAGUUCAAGUGGA 74 UCCACUUGAACUGGGUCAGAUCA 103XM_005264298.1_883-905_s 883 GGUCGUGUGAGGAAAGGGGCU 75AGCCCCUUUCCUCACACGACCAU 104 XM_005264298.1_675-697_s 675AUCGGAGCAGGUGAAGAUGCU 76 AGCAUCUUCACCUGCUCCGAUGC 105XM_005264298.1_800-822_s 800 ACGGAGACGUGGUGUUUGUCA 77UGACAAACACCACGUCUCCGUAG 106 XM_005264298.1_513-535_s 513GAUCCUGUGCGUGGGGCUAGU 78 ACUAGCCCCACGCACAGGAUCUG 107XM_005264298.1_875-897_s 875 GCUUGUAUGGUCGUGUGAGGA 79UCCUCACACGACCAUACAAGCCC 108 XM_005264298.1_796_818_s 796GGCUACGGAGACGUGGUGUUU 80 AAACACCACGUCUCCGUAGCCAA 109XM_005264298.1_891-913_s 891 GAGGAAAGGGGCUGUGCUUGU 81ACAAGCACAGCCCCUUUCCUCAC 110 XM_005264298.1_624-646_s 624GAAGGUUGAUCUGACCCAGUU 82 AACUGGGUCAGAUCAACCUUCUC 111XM_005264298.1_552-574_s 552 CCUGGUGGACAAGUACCCUAA 83UUAGGGUACUUGUCCACCAGGCU 112 XM_005264298.1_619-541_C21A_s 619UUUGAGAAGGUUGAUCUGACA 84 UGUCAGAUCAACCUUCUCAAAGU 113XM_005264298.1_628-650_G21A_s 628 GUUGAUCUGACCCAGUUCAAA 85UUUGAACUGGGUCAGAUCAACCU 114 XM_005264298.1_873-895_G21A_s 873GGGCUUGUAUGGUCGUGUGAA 86 UUCACACGACCAUACAAGCCCCU 115XM_005264298.1_836-858_G21A_s 836 AGCACUUGGGGUUCCAGUCAA 87UUGACUGGAACCCCAAGUGCUUG 116 XM_005264298.1_797-819_G21A_s 797GCUACGGAGACGUGGUGUUUA 88 UAAACACCACGUCUCCGUAGCCA 117XM_005264298.1_802-824_C21A_s 802 GGAGACGUGGUGUUUGUCAGA 89UCUGACAAACACCACGUCUCCGU 118

TABLE 5 Unmodified Sense and Antisense Strand Sequences of KHK dsRNAsSEQ SEQ Ref Seq; Corresponding mRNA Target ID IDposition of sense strand Site Sense (5′-3′) NO: Antisense (5′-3′) NO:XM_005264298.1_685-707_s 685 GUGAAGAUGCUGCAGCGGAUA 119UAUCCGCUGCAGCAUCUUCACCU 122 XM_005264298.1_687-709_s 687GAAGAUGCUGCAGCGGAUAGA 120 UCUAUCCGCUGCAGCAUCUUCAC 123XM_005264298.1_686-708_G21A_s 686 UGAAGAUGCUGCAGCGGAUAA 121UUAUCCGCUGCAGCAUCUUCACC 124

Example 3. Design, Synthesis, and in Vitro Screening of AdditionalsiRNAs

siRNA Design

An additional set of siRNAs targeting the human KHK, “ketohexokinase(fructokinase)” (human: NCBI refseqID XM_005264298; NCBI GeneID: 3795),as well as toxicology-species KHK orthologs (cynomolgus monkey:XM_005545463; mouse: NM_008439; rat, NM_031855) were designed usingcustom R and Python scripts. The human XM_005264298 REFSEQ mRNA has alength of 2146 bases. The rationale and method for the set of siRNAdesigns is as follows: the predicted efficacy for every potential 19mersiRNA from position 501 through position 2146 (the coding region and 3′UTR) was determined with a linear model derived from the direct measureof mRNA knockdown from more than 20,000 distinct siRNA designs targetinga large number of vertebrate genes. Subsets of the KHK siRNAs weredesigned with perfect or near-perfect matches between human, cynomolgusand rodent species as well as a subset targeting human and cynomolgusmonkey alone. A further subset was designed with perfect or near-perfectmatches to mouse and rat KHK orthologs. For each strand of the siRNA, acustom Python script was used in a brute force search to measure thenumber and positions of mismatches between the siRNA and all potentialalignments in the target species transcriptome. Extra weight was givento mismatches in the seed region, defined here as positions 2-9 of theantisense oligonucleotide, as well the cleavage site of the siRNA,defined here as positions 10-11 of the antisense oligonucleotide. Therelative weight of the mismatches was 2.8; 1.2:1 for seed mismatches,cleavage site, and other positions up through antisense position 19.Mismatches in the first position were ignored. A specificity score wascalculated for each strand by summing the value of each weightedmismatch. Preference was given to siRNAs whose antisense score in humanand cynomolgus monkey was >=3.0 and predicted efficacy was >=70%knockdown of the XM_005264298 transcript.

Synthesis

KHK siRNA sequences were synthesized at a 1 μmol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500° A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modifiednucleosides were introduced in the sequences using the correspondingphosphoramidites. Synthesis of 3′ GalNAc conjugated single strands wasperformed on a GalNAc modified CPG support. Custom CPG universal solidsupport was used for the synthesis of antisense single strands. Couplingtime for all phosphoramidites (100 mM in acetonitrile) was 5 minutesemploying 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M inacetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass.,USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3minutes. All sequences were synthesized with final removal of the DMTgroup (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagent at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 μL ofdimethyl sulfoxide (DMSO) and 300 μl TEA.3HF reagent was added and thesolution was incubated for additional 20 minutes at 60° C. At the end ofcleavage and deprotection step, the synthesis plate was allowed to cometo room temperature and was precipitated by addition of 1 mL ofacetontile: ethanol mixture (9:1). The plates were cooled at −80° C. for2 hours and the superanatant decanted carefully with the aid of a multichannel pipette. The oligonucleotide pellet was re-suspended in 20 mMNaOAc buffer and were desalted using a 5 mL HiTrap size exclusion column(GE Healthcare) on an AKTA Purifier System equipped with an A905autosampler and a Frac 950 fraction collector. Desalted samples werecollected in 96 well plates. Samples from each sequence were analyzed byLC-MS to confirm the identity, UV (260 nm) for quantification and aselected set of samples by IEX chromatography to determine purity.

Annealing of KHK single strands was performed on a Tecan liquid handlingrobot. Equimolar mixture of sense and antisense single strands werecombined and annealed in 96 well plates. After combining thecomplementary single strands, the 96 well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10 uM in 1×PBS and then submitted for in vitroscreening assays.

Cell Culture and Transfections

Hep3b cells were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl ofLipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. Forty μl of DMEMcontaining ˜5×10³ cells were then added to the siRNA mixture. Cells wereincubated for 24 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done over a range ofdoses from 10 nM to 36 fM final duplex concentration over 8, 6-folddilutions.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl ofLysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat 4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H2O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 h 37° C. Plates were thenincubated at 81° C. for 8 min.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDHTaqMan Probe (Hs99999905), 0.5 μl KHK probe (Hs00240827_m1) and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well plates (Roche cat #04887301001). Real time PCR was done in aLightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay.Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells.

A detailed list of KHK sense and antisense strand sequences is shown inTables 6 and 7.

The results of the single dose screens are provided in Table 8. Data areexpressed as percent message remaining relative to AD-1955.

Table 9 shows the dose response of a subset of agents in Hep3B cellstransfected with the indicated iRNAs. The indicated IC₅₀ valuesrepresent the IC₅₀ values relative to untreated cells.

TABLE 6 KHK Unmodified Sequences Ref Seq; SEQ Corresponding SEQCorresponding Duplex ID target mRNA ID target Name Sense strand NO:position Antisense strand NO: mRNA position AD-63824 AUCAAUGUGGUGGACAAAU125 NM_008439.3_ UUAUUUGUCCACCACAUUGAU 175 NM_008439.3_ AA 70-90_C21A_sGA 68-90_C21A_as AD-63829 GGUGGACAAAUACCCAGAG 126 NM_008439.3_UCCUCUGGGUAUUUGUCCACC 176 NM_008439.3_ GA 78-9880218_s AC 76-9880218_asAD-63855 CUUUGAGAAGGUCGAUCUG 127 NM_008439.3_ UUCAGAUCGACCUUCUCAAAG 177NM_008439.3_ AA 393-413_C21A_s UC 391-413_C21A_as AD-63835CCCGGUUCAAGUGGAUCCA 128 NM_008439.3_ UGUGGAUCCACUUGAACCGGG 178NM_008439.3_ CA 413-4335-213_s UC 411-4335-213_as AD-63845UGUGGCCAAGCACCUGGGG 129 NM_008439.3_ AACCCCAGGUGCUUGGCCACA 179NM_008439.3_ UU 603-6235-213_s UC 601-6235-213_as AD-63823UUGCAGGGGUUUGAUGGCA 130 NM_008439.3_ AAUGCCAUCAAACCCCUGCAA 180NM_008439.3_ UU 892-9124-212_s GC 890-9124-212_as AD-63863GAAGAGAAGCAGAUCCUGU 131 XM_005264298.1_ UCACAGGAUCUGCUUCUCUUC 181XM_005264298.1_ GA 504-524_C21A_s CA 502-524_C21A_as AD-63881AAGAGAAGCAGAUCCUGUG 132 XM_005264298.1_ UGCACAGGAUCUGCUUCUCUU 182XM_005264298.1_ CA 505-525_G21A_s CC 503-525_G21A_as AD-63862GAGAAGCAGAUCCUGUGCG 133 XM_005264298.1_ UACGCACAGGAUCUGCUUCUC 183XM_005264298.1_ UA 507-527_G21A_s UU 505-527_G21A_as AD-63887AGAAGCAGAUCCUGUGCGU 134 XM_005264298.1_ UCACGCACAGGAUCUGCUUCU 184XM_005264298.1_ GA 508-528_G21A_s CU 506-528_G21A_as AD-63902GAAGCAGAUCCUGUGCGUG 135 XM_005264298.1_ UCCACGCACAGGAUCUGCUUC 185XM_005264298.1_ GA 509-529_G21A_s UC 507-529_G21A_as AD-63896GCAGAUCCUGUGCGUGGGG 136 XM_005264298.1_ AGCCCCACGCACAGGAUCUGC 186XM_005264298.1_ CU 512-532_s UU 510-532_as AD-63843 GAUCCUGUGCGUGGGGCUA137 XM_005264298.1_ ACUAGCCCCACGCACAGGAUC 187 XM_005264298.1_ GU515-535_s UG 513-535_as AD-63857 GGUGGACAAGUACCCUAAG 138 XM_005264298.1_UCCUUAGGGUACUUGUCCACC 188 XM_005264298.1_ GA 557-577_s AG 555-577_asAD-63836 GCCUGCCAGAUGUGUCUGC 139 XM_005264298.1_ UAGCAGACACAUCUGGCAGGC189 XM_005264298.1_ UA 595-615_s UC 593-615_as AD-63834UUUGAGAAGGUUGAUCUGA 140 XM_005264298.1_ UGUCAGAUCAACCUUCUCAAA 190XM_005264298.1_ CA 621-641_C21A_s GU 619-641_C21A_as AD-63839GUUGAUCUGACCCAGUUCA 141 XM_005264298.1_ UUUGAACUGGGUCAGAUCAAC 191XM_005264298.1_ AA 630-650_G21A_s CU 628-650_G21A_as AD-63821AUCUGACCCAGUUCAAGUG 142 XM_005264298.1_ UCCACUUGAACUGGGUCAGAU 192XM_005264298.1_ GA 634-654_s CA 632-654_as AD-63847 CCAGUUCAAGUGGAUCCAC143 XM_005264298.1_ AUGUGGAUCCACUUGAACUGG 193 XM_005264298.1_ AU641-661_s GU 639-661_as AD-63846 CAGUUCAAGUGGAUCCACA 144 XM_005264298.1_AAUGUGGAUCCACUUGAACUG 194 XM_005264298.1_ UU 642-662_s GG 640-662_asAD-63826 AGUUCAAGUGGAUCCACAU 145 XM_005264298.1_ UAAUGUGGAUCCACUUGAACU195 XM_005264298.1_ UA 643-663_G21A_s GG 641-663_G21A_as AD-63841GUUCAAGUGGAUCCACAUU 146 XM_005264298.1_ UCAAUGUGGAUCCACUUGAAC 196XM_005264298.1_ GA 644-664_s UG 642-664_as AD-63856 UUCAAGUGGAUCCACAUUG147 XM_005264298.1_ UUCAAUGUGGAUCCACUUGAA 197 XM_005264298.1_ AA645-665_G21A_s CU 643-665_G21A_as AD-63868 UCAAGUGGAUCCACAUUGA 148XM_005264298.1_ UCUCAAUGUGGAUCCACUUGA 198 XM_005264298.1_ GA646-666_G21A_s AC 644-666_G21A_as AD-63869 CAAGUGGAUCCACAUUGAG 149XM_005264298.1_ UCCUCAAUGUGGAUCCACUUG 199 XM_005264298.1_ GA647-667_G21A_s AA 645-667_G21A_as AD-63880 AAGUGGAUCCACAUUGAGG 150XM_005264298.1_ UCCCUCAAUGUGGAUCCACUU 200 XM_005264298.1_ GA648-668_C21A_s GA 646-668_C21A_as AD-63875 AGUGGAUCCACAUUGAGGG 151XM_005264298.1_ UGCCCUCAAUGUGGAUCCACU 201 XM_005264298.1_ CA649-669_C21A_s UG 647-669_C21A_as AD-63874 GUGGAUCCACAUUGAGGGC 152XM_005264298.1_ UGGCCCUCAAUGUGGAUCCAC 202 XM_005264298.1_ CA650-670_G21A_s UU 648-670_G21A_as AD-63897 UGGAUCCACAUUGAGGGCC 153XM_005264298.1_ UCGGCCCUCAAUGUGGAUCCA 203 XM_005264298.1_ GA651-671_G21A_s CU 649-671_G21A_as AD-63879 GAUCCACAUUGAGGGCCGG 154XM_005264298.1_ UUCCGGCCCUCAAUGUGGAUC 204 XM_005264298.1_ AA 653-673_sCA 651-673_as AD-63819 GUGAAGAUGCUGCAGCGGA 155 XM_005264298.1_UAUCCGCUGCAGCAUCUUCAC 205 XM_005264298.1_ UA 687-707_s CU 685-707_asAD-63825 GAAGAUGCUGCAGCGGAUA 156 XM_005264298.1_ UCUAUCCGCUGCAGCAUCUUC206 XM_005264298.1_ GA 689-709_s AC 687-709_as AD-63837UGGCUACGGAGACGUGGUG 157 XM_005264298.1_ AACACCACGUCUCCGUAGCCA 207XM_005264298.1_ UU 797-817_s AA 795-817_as AD-63853 GGCUACGGAGACGUGGUGU158 XM_005264298.1_ AAACACCACGUCUCCGUAGCC 208 XM_005264298.1_ UU798-818_s AA 796-818_as AD-63854 GCUACGGAGACGUGGUGUU 159 XM_005264298.1_UAAACACCACGUCUCCGUAGC 209 XM_005264298.1_ UA 819_G21A_s CA797-819_G21A_as AD-63859 GGAGACGUGGUGUUUGUCA 160 XM_005264298.1_UCUGACAAACACCACGUCUCC 210 XM_005264298.1_ GA 824-C21A_s GU802-824_C21A_as AD-63820 ACGUGGUGUUUGUCAGCAA 161 XM_005264298.1_AUUUGCUGACAAACACCACGU 211 XM_005264298.1_ AU 808-828_G21U_s CU806-828_G21U_as AD-63851 GUGGUGUUUGUCAGCAAAG 162 XM_005264298.1_AUCUUUGCUGACAAACACCAC 212 XM_005264298.1_ AU 810-830_s GU 808-830_asAD-63873 GGUGUUUGUCAGCAAAGAU 163 XM_005264298.1_ ACAUCUUUGCUGACAAACACC213 XM_005264298.1_ GY 812-832_s AC 810-832_as AD-63886GUGUUUGUCAGCAAAGAUG 164 XM_005264298.1_ UACAUCUUUGCUGACAAACAC 214XM_005264298.1_ UA 813-833_G21A_s CA 811-833_G21A_as AD-63892UGUUUGUCAGCAAAGAUGU 165 XM_005264298.1_ UCACAUCUUUGCUGACAAACA 215XM_005264298.1_ GA 814-834_G21A_s CC 812-834_G21A_as AD-63901GUUUGUCAGCAAAGAUGUG 166 XM_005264298.1_ UCCACAUCUUUGCUGACAAAC 216XM_005264298.1_ GA 815-835_C21A_s AC 813-835_C21A_as AD-63885UUGUCAGCAAAGAUGUGGC 167 XM_005264298.1_ UGGCCACAUCUUUGCUGACAA 217XM_005264298.1_ CA 817-837_s AC 815-837_as AD-63861 UGUCAGCAAAGAUGUGGCC168 XM_005264298.1_ UUGGCCACAUCUUUGCUGACA 218 XM_005264298.1_ AA818-838_s AA 816-838_as AD-63842 UGUGGCCAAGCACUUGGGG 169 XM_005264298.1_AACCCCAAGUGCUUGGCCACA 219 XM_005264298.1_ UU 830-850_s UC 828-850_asAD-63849 AGCACUUGGGGUUCCAGUC 170 XM_005264298.1_ UUGACUGGAACCCCAAGUGCU220 XM_005264298.1_ AA 838-858_G21A_s UG 836-858_G21A_as AD-63844GGGCUUGUAUGGUCGUGUG 171 XM_005264298.1_ UUCACACGACCAUACAAGCCC 221XM_005264298.1_ AA 875-895_G21A_s CU 873-895_G21A_as AD-63832UUGUAUGGUCGUGUGAGGA 172 XM_005264298.1_ UUUCCUCACACGACCAUACAA 222XM_005264298.1_ AA 879-899_s GC 877-899_as AD-63827 GGUCGUGUGAGGAAAGGGG173 XM_005264298.1_ AGCCCCUUUCCUCACACGACC 223 XM_005264298.1_ CU885-905_s AU 883-905_as AD-63858 GAGGAAAGGGGCUGUGCUU 174 XM_005264298.1_ACAAGCACAGCCCCUUUCCUC 224 XM_005264298.1_ GU 893-913_s AC 891-913_as

TABLE 7 KHK Modified Sequences SEQ SEQ ID ID duplexName senseOligoNameSense strand NO: antisOligoName Antisense strand NO: Species AD-63824A-127677 AfsusCfaAfuGfuGfGf 225 A-127678 usUfsaUfuUfgUfcCfac 275 MmUfgGfaCfaAfaUfaAf cAfcAfuUfgAfusgsa L96 AD-63829 A-127663GfsgsUfgGfaCfaAfAf 226 A-127664 usCfscUfcUfgGfgUfau 276 MmUfaCfcCfaGfaGfgAf uUfgUfcCfaCfcsasc L96 AD-63855 A-127673CfsusUfuGfaGfaAfGf 227 A-127674 usUfscAfgAfuCfgAfcu 277 MmGfuCfgAfuCfuGfaAf uUfcUfcAfaAfgsusc L96 AD-63835 A0127665CfscsCfgGfuUfcAfAf 228 A-127666 usGfsuGfgAfuCfcAfcu 278 MmGfuGfgAfuCfcAfcAf uGfaAfcCfgGfgsusc L96 AD-63845 A-127669UfsgsUfgGfcCfaAfGf 229 A-127670 asAfscCfcCfaGfgUfgc 279 MmCfaCfcUfgGfgGfuUf uUfgGfcCfaCfasusc L96 AD-63823 A-127661UfsusGfcAfgGfgGfUf 230 A-127662 asAfsuGfcCfaUfcAfaa 280 MmUfuGfaUfgGfcAfuUf cCfcCfuGfcAfasgsc L96 AD-63863 A-127587GfsasAfgAfgAfaGfCf 231 A-127588 usCfsaCfaGfgAfuCfug 281 HsAfgAfuCfcUfgUfgAf cUfuCfuCfuUfcscsa L96 AD-63881 A-127593AfsasGfaGfaAfgCfAf 232 A-127594 usFfscAfcAfgGfaUfcu 282 HsGfaUfcCfuGfuGfcAf gCfuUfcUfcUfuscsc L96 AD-63862 A-127571GfsasGfaAfgCfaGfAf 233 A-127572 usAfscGfcAfcAfgGfau 283 HsUfcCfuGfuGfcGfuAf cUfgCfuUfcUfcsusu L96 AD-63887 A-127595AfsgsAfaGfcAfgAfUf 234 A-127596 usCfsaCfgCfaCfaGfga 284 HsCfcUfgUfgCfgUfgAf uCfuGfcUfuCfuscsu L96 AD-63902 A-127585GfsasAfgCfaGfaUfCf 235 A-127586 usCfscAfcGfcAfcAfgg 285 HsCfuGfuGfcGfuGfgAf aUfcUfgCfuUfcsusc L96 AD-63896 A-127567GfscsAfgAfuCfcUfGf 236 A-127568 asGfscCfcCfaCfgCfac 286 HsUfgCfgUfgGfgGfcUf agFgAfuCfuGfcsusu L96 AD-63843 A-127637GfsasUfcCfuGfuGfCf 237 A-127638 asCfsuAfgCfcCfcAfcg 287 HsGfuGfgGfgCfuAfgUf cAfcAfgGfaUfcsusg L96 AD-63857 A-127627GfsgsUfgGfaCfaAfGf 238 A-127628 usCfscUfuAfgGfgUfac 288 HsUfaCfcCfuAfaGfgAf uUfgUfcCfaCfcsasg L96 AD-63836 A-127603GfscsCfuGfcCfaGfAf 239 A-127604 usAfsgCfaGfaCfaCfau 289 HsUfgUfgUfcUfgCfuAf cUfgGfcAfgGfcsusc L96 AD-63834 A-127649UfsusUfgAfgAfaGfGf 240 A-127650 usGfsuCfaGfaUfcAfac 290 HsUfuGfaUfcUfgAfcAf cUfuCfuCfaAfasgsu L96 AD-63839 A-127651GfsusUfgAfuCfuGfAf 241 A-127652 usUfsuGfaAfcUfgGfgu 291 HsCfcCfaGfuUfcAfaAf cAfgAfuCfaAfcscsu L96 AD-63821 A-127629AfsusCfuGfaCfcCfAf 242 A-127630 usCfscAfcUfuGfaAfcu 292 HsGfuUfcAfaGfuGfgAf gGfgUfcAfgAfuscsa L96 AD-63847 A-127623CfscsAfgUfuCfaAfGu 243 A-127624 asUfsgUfgGfaUfcCfac 293 HsUfgGfaUfcCfaCfaUf uUfgAfaCfuGfgsgsu L96 AD-63846 A-127607CfsasGfuUfcAfaGfUf 244 A-127608 asAfsuGfuGfgAfuCfca 294 HsGfgAfuCfcAfcAfuUf cUfuGfaAfcUfgsgsg L96 AD-63826 A-127615AfsgsUfuCfaAfgUfGf 245 A-127616 usAfsaUfgUfgGfaUfcc 295 HsGfaUfcCfaCfaUfuAf aCfuUfgAfaCfusgsg L96 AD-63841 A-127605GfsusUfcAfaGfuGfGf 246 A-127606 usCfsaAfuGfuGfgAfuc 296 HsAfuCfcAfcAfuUfgAf cAfcUfuGfaAfcsusg L96 AD-63856 A-127611UfsusCfaAfgUfgGfAf 247 A-127612 usUfscAfaUfgUfgGfau 297 HsUfcCfaCfaUfuGfaAf cCfaCfuUfgAfascsu L96 AD-63867 A-127573UfscsAfaGfuGfgAfUf 248 A-127574 usCfsuCfaAfuGfuGfga 298 HsCfcAfcAfuUfgAfgAf uCfcAfcUfuGfasasc L96 AD-63869 A-127589CfsasAfgUfgGfaUfCf 249 A-127590 usCfscUfcAfaUfgUfgg 299 HsCfaCfaUfuGfaGfgAf aUfcCfaCfuUfgsasa L96 AD-63880 A-127577AfsasGfuGfgAfuCfCf 250 A-127578 usCfscCfuCfaAfuGfug 300 HsAfcAfuUfgAfgGfgAf gAfuCfcAfcUfusgsa L96 AD-63875 A-127591AfsgsUfgGfaUfcCfAf 251 A-127592 usGfscCfcUfcAfaUfgu 301 HsCfaUfuGfaGfgGfcAf gGfaUfcCfaCfususg L96 AD-63874 A-127575GfsusGfgAfuCfcAfCf 252 A-127576 usGfsgCfcCfuCfaAfug 302 HsAfuUfgAfgGfgCfcAf uGfgAfuCfcAfcsusu L96 AD-63897 A-127583UfsgsGfaUfcCfaCfAf 253 A-127584 usCfsgGfcCfcUfcAfau 303 HsUfuGfaGfgGfcCfgAf gUfgGfaUfcCfascsu L96 AD-63879 A-127561GfsasUfcCfaCfaUfUf 254 A-127562 usUfscCfgGfcCfcUfca 304 HsGfaGfgGfcCfgGfaAf aUfgUfgGfaUfcscsa L96 AD-63819 A-127597GfsusGfaAfgAfuGfCf 255 A-127598 usAfsuCfcGfcUfgCfag 305 HsUfgCfaGfcGfgAfuAf cAfuCfuUfcAfcscsu L96 AD-63825 A-127599GfsasAfgAfuGfcUfGf 256 A-127600 usCfsuAfuCfcGfcUfgc 306 HsCfaGfcGfgAfuAfgAf aGfcAfuCfuUfcsasc L96 AD-63837 A-127619UfsgsGfcUfaCfgFfAf 257 A-127620 asAfscAfcCfaCfgUfcu 307 HsGfaCfgUfgGfuGfuUf cCfgUfaGfcCgasasa L96 AD-63853 A-127641GfsgsCfuAfcGfgAfGf 258 A-127642 asAfsaCfaCfcAfcGfuc 308 HsAfcGfuGfgUfgUfuUf uCfcGfuAfgCfcsasa L96 AD-63854 A-127657GfscsUfaCfgGfaGfAf 259 A-127658 usAfsaAfcAfcCfaCfgu 309 HsCfgUfgGfuGfuUfuAf cUfcCfgUfaGfcscsa L96 AD-63859 A-127659GfsgsAfgAfcGfuGfGf 260 A-127660 usCfsuGfaCfaAfaCfac 310 HsUfgUfuUfgUfcAfgAf cAfcGfuCfuCgcsgsu L96 AD-63820 A-127613AfscsGfuGfgUfgUfUf 261 A-127614 asUfsuUfgCfuGfaCfaa 311 HsUfgUfcAfgCfaAfaUf aCfaCfcAfcGfuscsu L96 AD-63851 A-127609GfsusGfgUfgUfuUfGu 262 A-127640 asUfscUfuUfgCfuGfac 312 HsUfcAfgCfaAfaGfaUf aAfaCfaCfcAfcsgsu L96 AD-63873 A-127559GfsgsUfgUfuUfgUfCf 263 A-127560 asCfsaUfcUfuUfgCfug 313 HsAfgCfaAfaGfaUfgUf aCfaAfaCfaCfcsasc L96 AD-63886 A-127579GfsusGfuUfuGfuCfAf 264 A-127580 usAfscAfuCfuUfuGfcu 314 HsGfcAfaAfgAfuGfuAf gAfcAfaAfcAfcscsa L96 AD-63892 A-127581UfsgsUfuUfgUfcAfGf 265 A-127582 usCfsaCfaUfcUfuUfgc 315 HsCfaAfaGfaUfgUfgAf uGfaCfaAfaCfascsc L96 AD-63901 A-127569GfsusUfuGfuCfaGfCf 266 A-127570 usCfscAfcAfuCfuUfug 316 HsAfaAfgAfuGfuGfgAf cUfgAfcAfaAfcsasc L96 AD-63885 A-127563UfsusGfuCfaGfcAfAf 267 A-127564 usGfsgCfcAfcAfuCfuu 317 HsAfgAfuGfuGfgCfcAf uGfcUfgAfcAfasasc L96 AD-63861 A-127555UfsgsUfcAfgCfaAfAf 268 A-127556 usUfsgGfcCfaCfaUfcu 318 HsGfaUfgUfgGfcCfaAf uUfgCfuGfaCfasasa L96 AD-63842 A-127621UfsgsUfgGfcCfaAfGf 269 A-127622 asAfscCfcCfaAfgUfgc 319 HsCfaCfuUfgGfgGfuUf uUfgGfcCfaCfasusc L96 AD-63849 A-127655AfsgsCfaCfuUfgGfGf 270 A-127656 usUfsgAfcUfgGfaAfcc 320 HsGfuUfcCfaGfuCfaAf cCfaAfgUfgCfususg L96 AD-63844 A-127653GfsgsGfcUfuGfuAfUf 271 A-127654 usUfscAfcAfcGfaCfca 321 HsGfgUfcGfuGfuGfaAf uAfcAfaGgcCfcscsu L96 AD-63832 A-127617UfsusGfuAfuGfgUfCf 272 A-127618 usUfsuCfcUfcAfcAfcg 322 HsGfuGfuGfaGfgAfaAf aCfcAfuAfcAfasgsc L96 AD-63827 A-127631GfsgsUfcGfuGfuGfAf 273 A-127632 asGfscCfcCfuUfuCfcu 323 HsGfgAfaAfgGfgGfcUf cAfcAfcGfaCfcsasu L96 AD-63858 A-127643GfsasGfgAfaAfgGfGf 274 A-127644 asCfsaAfgCfaCfaGfcc 324 HsGfcUfgUfgCfuUfgUf cCfuUfuCfcUfcsasc L96

TABLE 8 KHK Single Dose Screen in Hep3b 10 0.1 10 0.1 DuplexID nM_AVGnM_AVG nM_STDEV nM_STDEV AD-63819 82.0 82.1 24.1 10.0 AD-63820 11.2 45.6N/A 3.6 AD-63821 68.2 102.4 19.8 10.5 AD-63823 134.3 84.4 22.3 14.0AD-63824 94.1 98.0 4.1 4.3 AD-63825 70.4 77.7 6.5 5.3 AD-63826 45.3 89.75.5 7.5 AD-63827 51.7 130.0 21.2 82.8 AD-63829 89.6 78.2 1.3 1.5AD-63832 17.4 80.5 1.4 19.5 AD-63834 11.0 72.7 0.8 20.7 AD-63835 17.2103.5 8.1 76.4 AD-63836 37.7 63.8 2.8 23.5 AD-63837 87.3 66.8 33.0 5.2AD-63839 N/A 34.0 N/A 2.8 AD-63841 46.4 64.4 0.7 7.9 AD-63842 72.6 76.922.7 13.9 AD-63843 48.6 108.0 9.0 35.8 AD-63844 68.2 80.1 16.5 12.9AD-63845 54.4 68.1 43.3 4.0 AD-63846 66.0 88.9 18.8 2.2 AD-63847 43.758.1 17.5 12.7 AD-63849 31.0 85.4 4.4 30.3 AD-63851 28.9 29.5 4.1 21.4AD-63853 39.9 49.3 0.0 11.7 AD-63854 7.1 53.3 2.6 10.1 AD-63855 15.032.6 14.5 7.3 AD-63856 66.8 96.9 3.3 15.1 AD-63857 64.9 101.4 7.3 3.5AD-63858 67.5 89.9 5.6 18.8 AD-63859 44.8 91.0 1.1 10.2 AD-63861 78.289.1 0.0 8.3 AD-63862 48.0 92.9 14.8 14.5 AD-63863 39.3 65.7 17.9 13.1AD-63868 47.4 106.8 4.4 13.1 AD-63869 20.7 68.0 3.8 28.2 AD-63873 22.6102.8 8.0 31.7 AD-63874 64.7 107.6 2.9 6.3 AD-63875 85.2 108.4 30.6 60.3AD-63879 66.1 81.1 3.6 7.9 AD-63880 86.7 85.9 19.4 3.3 AD-63881 60.863.1 4.5 3.1 AD-63885 75.1 66.3 12.4 11.6 AD-63886 27.5 53.3 5.1 7.8AD-63887 40.0 70.5 8.4 0.7 AD-63892 19.9 82.7 1.5 5.7 AD-63896 88.6 36.61.7 15.6 AD-63897 96.7 91.4 4.3 4.9 AD-63901 48.1 97.6 4.5 2.9 AD-6390243.1 99.2 8.6 9.7

TABLE 9 KHK Dose Response Screen in Hep3b Duplex ID IC50 (nM) AD-638510.027 AD-63820 0.010 AD-63853 0.067 AD-63839 0.015 AD-63854 0.075AD-63855 0.017853 AD-63885 0.082879

1. A double stranded RNAi agent for inhibiting expression of aketohexokinase (KHK) gene, wherein the double stranded RNAi agent isselected from the group consisting (a) a double stranded RNAi agent forinhibiting expression of a ketohexokinase (KHK) gene, wherein the doublestranded RNAi agent comprises a sense strand and an antisense strandforming a double stranded region, wherein said sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:1, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2; (b) a doublestranded RNAi agent for inhibiting expression of a ketohexokinase (KHK)gene, wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, the antisensestrand comprising a region of complementarity which comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the antisense sequences listed in any one of Tables 3, 4, 5,6, and 7; (c) a double stranded RNAi agent for inhibiting expression ofa ketohexokinase (KHK) gene, wherein said double stranded RNAi agentcomprises a sense strand and an antisense strand forming adouble-stranded region, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1, and said antisense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2. whereinsubstantially all of the nucleotides of said sense strand andsubstantially all of the nucleotides of said antisense strand aremodified nucleotides, and wherein said sense strand is conjugated to aligand attached at the 3′-terminus; (d) a double stranded RNAi agentcapable of inhibiting the expression of a ketohexokinase (KHK) gene,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region, wherein saidantisense strand comprises a region complementary to part of an mRNAencoding KHK, wherein each strand is about 14 to about 30 nucleotides inlength, wherein said double stranded RNAi agent is represented byformula (III): sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: j, k, and l are each independently 0 or 1; p, p′, q, and q′ areeach independently 0-6; each N_(a) and N_(a)′ independently representsan oligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides; each N_(b) and N_(b)′independently represents an oligonucleotide sequence comprising 0-10nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present, independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; and whereinthe sense strand is conjugated to at least one ligand; and (e) a doublestranded RNAi agent for inhibiting expression of a ketohexokinase (KHK),gene wherein said double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, wherein saidsense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1,and said antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:2, wherein substantially all of the nucleotides of said sensestrand comprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification, wherein saidsense strand comprises two phosphorothioate internucleotide linkages atthe 5′-terminus, wherein substantially all of the nucleotides of saidantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein said antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein said sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.
 2. Thedouble stranded RNAi agent of claim 1, wherein said double stranded RNAiagent comprises at least one modified nucleotide.
 3. (canceled)
 4. Thedouble stranded RNAi agent of claim 1, further comprising a ligand. 5.The double stranded RNAi agent of claim 1, wherein i is 0; j is 0; i is1; j is 1; both i and j are 0; or both i and j are 1; or wherein k is 0;l is 0; k is 1; l is 1; both k and l are 0; or both k and l are
 1. 6.(canceled)
 7. The double stranded RNAi agent of claim 1, wherein XXX iscomplementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ iscomplementary to Z′Z′Z′.
 8. The double stranded RNAi agent of claim 1,wherein the YYY motif occurs at or near the cleavage site of the sensestrand.
 9. The double stranded RNAi agent of claim 1, wherein formula(III) is represented by formula (IIIa): sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′ (IIIa).;

or wherein formula (III) is represented by formula (IIIb): sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′ (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides; or whereinformula (III) is represented by formula (IIIc): sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′  antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(a′)-n_(q′) 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides; or whereinformula (III) is represented by formula (IIId): sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′ (IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.
 10. The double stranded RNAi agentof claim 1, wherein the double-stranded region is 15-30 nucleotide pairsin length.
 11. The double stranded RNAi agent of claim 1, wherein eachstrand has 15-30 nucleotides.
 12. The double stranded RNAi agent ofclaim 1, wherein the ligand is


13. The double stranded RNAi agent of claim 1, wherein the ligand isattached to the 3′ end of the sense strand.
 14. The double stranded RNAiagent of claim 13, wherein the RNAi agent is conjugated to the ligand asshown in the following schematic

wherein X is O or S.
 15. The double stranded RNAi agent of claim 1,wherein said RNAi agent further comprises at least one phosphorothioateor methylphosphonate internucleotide linkage.
 16. (canceled) 17.(canceled)
 18. A cell containing the double stranded RNAi agent ofclaim
 1. 19. A pharmaceutical composition comprising the double strandedRNAi agent of claim
 1. 20. A method of inhibiting ketohexokinase (KHK)expression in a cell, the method comprising: (a) contacting the cellwith the double stranded RNAi agent of claim 1; and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of a KHK gene, thereby inhibiting expression of theKHK gene in the cell. 21-23. (canceled)
 24. A method of treating asubject having a ketohexokinase (KHK)-associated disorder, comprisingsubcutaneously administering to the subject a therapeutically effectiveamount of a double stranded RNAi agent of claim 1, thereby treating thesubject.
 25. The method of claim 24, wherein the subject is a human. 26.The method of claim 24, wherein the ketohexokinase-associated disease isselected from the group consisting of liver disease, dyslipidemia,disorders of glycemic control, cardiovascular disease, kidney disease,metabolic syndrome, adipocyte dysfunction, visceral adipose deposition,obesity, hyperuricemia, gout, eating disorders, and excessive sugarcraving.
 27. The method of claim 24, wherein the double stranded RNAiagent is administered to the subject at a dose of about 0.01 mg/kg toabout 10 mg/kg; or at a dose of about 1 mg/kg to about 10 mg/kg.
 28. Themethod of claim 24, wherein the double stranded RNAi agent isadministered to the subject subcutaneously; or the double stranded RNAiagent is administered to the subject intravenously.
 29. (canceled) 30.(canceled)